Shared developmental roles and transcriptional control of
autophagy and apoptosis in Caenorhabditis elegans
PhD dissertation
Péter Erdélyi
Eötvös Loránd University, Faculty of Science
Doctoral School of Biology
Classical and Molecular Genetics PhD Program
Leader of the PhD School: Anna Erdei DSc, member of HAS
Leader of PhD Program: László Orosz DSc, member of HAS
Supervisor: Krisztina Takács –Vellai PhD, habilitated assistant professor
Eötvös Loránd University, Faculty of Science
Department of Genetics
Budapest
2012
Table of Contents
List of abbreviations……………………………………………………………………………………………………………………………5
Chapter I…………………………………………………………………………………………………………………………………………..10
1
General introduction ..................................................................................................................... 11
1.1
1.1.1
Introduction ................................................................................................................... 11
1.1.2
The development of our knowledge about PCD ........................................................... 11
1.1.3
Morphological features of PCDs .................................................................................... 12
1.2
3
Apoptosis ............................................................................................................................... 13
1.2.1
Introduction ................................................................................................................... 13
1.2.2
The mechanism of apoptosis in C. elegans ................................................................... 13
1.2.3
The mechanism of apoptosis in mammals .................................................................... 18
1.2.4
Regulation of Apoptosis ................................................................................................ 20
1.2.5
The functions of apoptosis ............................................................................................ 24
1.3
2
Programmed cell death in animal development ................................................................... 11
Autophagy ............................................................................................................................. 26
1.3.1
Introduction ................................................................................................................... 26
1.3.2
The mechanism of autophagy ....................................................................................... 27
1.3.3
The regulation of autophagy ......................................................................................... 35
1.3.4
Functions of autophagy in animal development ........................................................... 38
1.3.5
Autophagy in C. elegans ................................................................................................ 41
Aims ............................................................................................................................................... 43
2.1
Investigating simultaneous inactivation of autophagy and apoptosis in C. elegans ............ 43
2.2
Common regulation of autophagy and apoptosis ................................................................. 44
Materials and methods ................................................................................................................. 45
3.1
Maintenance of C. elegans strains ........................................................................................ 45
3.2
Genetic crossing, generating double mutants ...................................................................... 45
3.3
RNAi treatment...................................................................................................................... 47
3.4
Nematode strains and RNAi constructs ................................................................................ 48
3.5
Generated strains .................................................................................................................. 49
3.6
Determining lethality and brood size .................................................................................... 49
3.7
Bioinformatics and statistical analysis................................................................................... 50
3.8
Light and fluorescence microscopy ....................................................................................... 50
3.9
Measuring the level of apoptosis .......................................................................................... 51
2
4
3.10
EMSA (electrophoretic mobility shift assay) ......................................................................... 52
3.11
Cloning of reporter constructs .............................................................................................. 52
3.12
Generation of transgenic animals (microparticle bombardment) ........................................ 53
Results ........................................................................................................................................... 54
4.1
Autophagy and apoptosis are redundantly required for C. elegans development............... 54
4.1.1
Mutational inactivation of atg-18 in apoptosis deficient genetic background causes full
penetrant embryonic lethality ...................................................................................................... 54
4.1.2
Inactivation of ced-4 enhances the embryonic and larval lethality of unc-51 mutants 57
4.1.3
Inactivation of atg-7 and lgg-1 in ced-4 loss-of-function mutant background causes
embryonic lethality........................................................................................................................ 61
4.1.4
Silencing of bec-1 decreases viability and causes sterility in apoptosis deficient
mutants 63
4.1.5
Simultaneous inactivation of autophagy and apoptosis causes a synthetic brood sizereduction phenotype in C. elegans................................................................................................ 65
4.1.6
The level of apoptosis increases in autophagic mutant background ............................ 66
4.1.7
Autophagic activity does not change in apoptotic mutant background ....................... 68
4.2
5
Autophagy and apoptosis are transcriptionally co-regulated in C. elegans.......................... 70
4.2.1
The promoter region of bec-1 contains a CES-2/ATF-2 binding site ............................. 70
4.2.2
CES-2 and ATF-2 can bind to the promoter region of bec-1 in vitro ............................. 71
4.2.3
The bZip-like binding site is crucial in the regulation of bec-1 ...................................... 73
Discussion ...................................................................................................................................... 77
5.1
Interplay between apoptosis and autophagy ....................................................................... 77
5.2
Autophagy and apoptosis are necessary together for the viability of C. elegans ................. 78
5.3
Simultaneous inactivation of autophagy and apoptosis causes severe morphological
malformations during embryogenesis .............................................................................................. 81
5.4
Autophagy and apoptosis are redundantly required for the embryonic development of C.
elegans .............................................................................................................................................. 83
6
5.5
Autophagy and apoptosis together have an influence on the fertility of C. elegans............ 84
5.6
The role of bec-1 in the regulation of autophagy and apoptosis .......................................... 85
References ..................................................................................................................................... 88
Chapter II………………………………………………………………………………………………………………………………………..107
7
General introduction ................................................................................................................... 108
7.1
The muscle structure of C. elegans ..................................................................................... 108
7.2
The myosin .......................................................................................................................... 110
8
Aims ............................................................................................................................................. 112
9
Matherials and Methods ............................................................................................................. 113
3
9.1
C. elegans motility assay ..................................................................................................... 113
9.2
Determining brood size and the number of bags of worms ............................................... 113
9.3
Lifespan assay of C. elegans ................................................................................................ 113
9.4
Used and generated strains................................................................................................. 114
10
Results ..................................................................................................................................... 115
11
Discussion ................................................................................................................................ 121
12
References ............................................................................................................................... 123
13
Summary.................................................................................................................................. 125
14
Összefoglalás ........................................................................................................................... 126
15
Acknowledgements ................................................................................................................. 127
4
List of Abbreviations
A band -
anisotropic - a part of the sarcomere
ACD -
autophagic cell death
AMP -
adenosine monophosphate
AMPK -
AMP-activated protein kinase
Apaf -
apoptosis activating factor
ARMS-PCR -
amplification refractory mutation system polymerase chain reaction
ARTS -
apoptosis-related protein in the TGF-β signalling pathway
arx -
ARp2/3 compleX component
ATF -
cAMP-dependent transcription factor
Atg -
autophagy-related
ADP -
adenosine diphosphate
ATP -
adenosine triphosphate
Bad -
Bcl-2-associated agonist of cell death
BAK -
Bcl-2 homologous antagonist killer
Bax -
Bcl-2-associated X protein
Bcl -
B cell lymphoma
bec -
beclin homolog
BECN -
beclin
BH3 -
Bcl-2 homology domain 3
BH4 -
Bcl-2 homology domain 4
bHLH -
basic helix-loop-helix
Bid -
BH3 interacting domain death agonist
BIR -
baculovirus inhibitory repeat
BL -
basal lamina
BNIP3 -
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
BOK -
Bcl-2-related ovarien killer
bZip -
basic leucine zipper
cAMP -
cyclic adenosine monophosphate
CD -
cluster of diffrentiation
ced -
cell death abnormality
5
CES -
cell death specification
clp -
calpain family
CMA -
chaperone-mediated autophagy
cps -
CED-3 protease supressor
crn -
cell death related nucleases
CXCL -
chemokine ligand
cyp -
cyclophilins
csp -
caspase
daf -
abnormal dauer formation
DAPI -
4',6-diamidino-2-phenylindole
DB -
dense body
Diap -
Drosophila inhibitor of apoptosis
DIC -
differential interference contrast
DIF -
differentiation factor
Dpp -
decapentaplegic
dUTP -
deoxyuridine triphosphate
EDTA -
ethylenediaminetetraacetic acid
egl -
egg-laying defective
EMSA -
electrophoretic mobility shift assay
epg -
ectopic P granules
ERK -
extracellular signal-regulated kinase
evl -
abnormal eversion of vulva
FIP200 -
focal adhesion kinase family interacting protein of 200 kD
FoxO -
forkhead box
FUdR -
5-fluorodeoxyuridine [5-Fluoro-1-[4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidine-2,4-dione]
GABARAP -
GABA(A) receptor-associated protein
GFP -
green fluorescent protein
GTPase -
guanosine triphosphatase
H band -
Heller band
HEK293 -
human embryonic kidney 293 cell line
Hox -
homeobox
6
HSN -
hermaphrodite-specific neurons
hVPS -
human vacuolar protein sorting
I band -
isotropic - a part of the sarcomere
IAP -
inhibitor of apoptosis
IBM -
IAP-binding motif
icd -
inhibitor of cell death
IGF -
insulin-like growth factor
IGFR -
insulin-like growth factor receptor
IPTG -
isopropyl β-D-1-thiogalactopyranoside
JNK -
jun N-terminal kinase
LAMP -
lysosomal-associated membrane protein
LB -
Luria-Bertani medium
LC3 -
microtubule-associated protein 1 light chain 3
let -
lethal
lgg -
LC3, GABARAP and GATE-16 family
lin -
abnormal cell lineage
lon -
long
LPC -
lysophospatidylcholine
M line -
middle line in sarcomere
MAP1LC3A - microtubule-associated protein 1 light chain 3 alpha
MEF -
mouse embryonic fibroblast
MEK -
MAPK/ERK kinase
MHC-B -
myosin heavy chain B
MIG -
abnormal cell migration
MOM -
mitochondrial outer membrane
MOMP -
mitochondrial outer membrane permeabilization
myo -
myosin heavy chain structural genes
NGM -
nematode growth medium
NSM -
neuro-secretory motorneuron
nuc -
nuclease defective
PAS -
pre-autophagosomal structure
PBS -
phosphate buffered saline
7
PCD -
programmed cell death
PDK -
phosphoinositide-dependent protein kinase
PE -
phosphatidylethanolamine
PIK3C3 -
phosphoinositide-3-kinase, class 3
PKB -
protein kinase B
PS -
phosphatidylserine
psr -
phosphatidylserine receptor family
PtdIns(3)P -
phosphatidylinositol (3)-phosphate
PtdIns3K -
phosphatidylinositol 3-kinase
Rac -
ras-related C3 botulinum toxin substrate
Raptor -
regulatory associated protein of mTORC
Rheb -
Ras homolog enriched in brain
RHG -
reaper, hid, grim
RNAi -
RNA interference
ROS -
reactive oxygen species
Rubicon -
RUN domain and cysteine-rich domain containing, Beclin1-interacting
S1P -
sphingosine 1 phosphate
Sirt -
sirtuin
SNARE -
soluble NSF attachment protein receptor
SWPCR -
single worm polymerase chain reaction
TBE-PAGE -
Tris/Borate/EDTA polyacrilamide gele electrophoresis
TdT -
terminal deoxynucleotidyl transferase
TOR -
target of rapamycin
TORC -
target of rapamycin complex
tra -
transformer
TSC -
tuberous sclerosis complex
TUNEL -
terminal deoxynucleotidyl transferase dUTP nick end labeling
TXRED -
texas red
ULK -
unc-51-like kinase
unc -
uncoordinated
UVRAG -
UV radiation resistance associated gene
Vam -
vacuolar morphogenesis
8
VMP -
vacuole membrane protein
Vps -
vacuolar sorting protein
Vti -
Vps10 (ten) interacting
wah -
worm AIF homolog
WDR45L -
WD repeat domain 45 light
WIPI -
WD repeat domain, phosphoinositide interacting
wsp -
WASP (actin cytoskeleton modulator) homolog
XIAP -
X-linked inhibitor of apoptosis
Ypt -
yeast protein two
Z disc -
Zwischenscheibe - a part of the sarcomere
9
Chapter I.
Shared developmental roles and transcriptional control of
autophagy and apoptosis in Caenorhabditis elegans
10
1 General introduction
1.1 Programmed cell death in animal development
1.1.1 Introduction
Programmed cell death (PCD) is essential in animal development and tissue
homeostasis. There are three major types of PCD: apoptosis (type I PCD), autophagy (type II
PCD) and necrosis (type III PCD), although necrosis is not always considered as programmed
cell death. PCD functions during animal development to eliminate certain cells, tissues or
complete organs, for example larval structures in insects. It helps in the formation of
developing tissues, regulates cell number and eliminates dangerous cells. Abnormal function
of PCD can lead to several diseases in human, including neurodegeneration, immunological
and developmental disorders or cancer (Fuchs and Steller, 2011).
1.1.2 The development of our knowledge about PCD
Cell death was already observed in the 19th century, and it was considered as a
passive phenomenon, an inevitable fate for cells (Glücksmann, 1951). Later, investigations of
developmentally regulated cell death in silkworm and tadpole began to change this view. It
was shown that inhibitors of protein or RNA synthesis can delay cell death and that survival
of neurons requires extracellular factors termed neurotrophins (Lockshin and Williams,
1965; Tata, 1966). It became clear later that these factors suppress the execution of an
intrinsic cell suicide program and this mechanism is not restricted to the nervous system. It
has been discovered that competition for limiting survival signals is a widely used general
mechanism that regulates cell number in animals (Jacobson et al., 1997). The next step in
our understanding of cell death came from an ultrastructural study performed by Currie and
coworkers, who described a series of distinct morphological changes in cells dying under
physiological conditions (Kerr et al., 1972). Cells that die during normal development shrink,
have condensed nuclei, retain membrane integrity, and are rapidly eliminated by
phagocytosis in a process called apoptosis (Jacobson et al., 1997). More recent studies have
uncovered other forms of programmed cell death, for example autophagy, revealing that
apoptosis is not the only form of developmental cell elimination and that additional
mechanisms likely compensate when it is prevented (Yuan and Kroemer, 2010).
11
Genetic studies on the nematode Caenorhabditis elegans provided a breakthrough
in understanding the mechanism of PCD. The identification of genetic pathways that
specifically affect programmed cell death demonstrated that cell suicide is developmentally
programmed, and specific genes are responsible for its initiation and execution (Metzstein et
al., 1998; Ellis and Horvitz, 1986). These studies led to the characterization of an
evolutionary conserved core cell death machinery. A family of cysteine proteases, termed
caspases, constitutes the downstream components of this machinery, as the key executors
of apoptosis (Hengartner, 2000; Thornberry and Lazebnik, 1998). Our knowledge about the
role and regulation of PCD arose primarily from three model systems, the nematode C.
elegans, the fruit fly Drosophila melanogaster, and the rodent Mus musculus (Fuchs and
Steller, 2011).
1.1.3 Morphological features of PCDs
1.1.3.1 Apoptosis
Apoptosis, also known as type I cell death, is characterised by rounding up of the
cell, retraction of pseudopods, reduction of cellular volume (pyknosis), chromatin
condensation, nuclear fragmentation (karyorrhexis), few or no ultrastructural modifications
of cytoplasmic organelles, and plasma membrane blebbing (Fig. 1). However, the integrity of
the affected cell is maintained until it is eliminated by phagocytosis. The term apoptosis
should be applied to cell death events which display these morphological features (Kroemer
et al., 2005). There are distinct subtypes of apoptosis, for example it can be regulated
through the intrinsic or the extrinsic pathway, and apoptosis can be processed with or
without caspase activation, they are morphologically similar. Although this process is the
best known type, PCD is not synonymous with apoptosis, as PCD can occur with nonapoptotic features as well.
1.1.3.2 Autophagic cell death
Autophagic cell death (ACD), also called type II cell death, is accompanied by largescale autophagic vacuolization of the cytoplasm and the absence of chromatin condensation
(Kroemer and Levine, 2008) (Fig. 1). There are evidences that ACD eliminates cells alone,
however, under several conditions these morphological features occure together with
apoptosis or necrosis (Kroemer and Levine, 2008).
12
1.1.3.3 Necrotic cell death or necrosis
Necrotic cell death or necrosis, also known as type III cell death, is morphologically
characterized by a gain in cell volume (oncosis), swelling of organelles and plasma
membrane rupture and subsequent loss of intracellular contents (Fig. 1). Conventionally,
necrosis is known as an accidental, uncontrolled form of cell death that only belongs to
pathological occurrences. However, evidence is accumulating that necrotic cell death is
regulated by several signal transduction pathways (Festjens et al., 2006). Furthermore, to
separate regulated necrosis from accidental events, the term ‘necroptosis’ has been created.
Figure 1. Electron microscopy images showing the morphology of different types of cell death Cells dying by apoptosis (a), autophagic cell death (b) or necrosis (c) show characteristic and unique
morphological features (Kroemer and Levine, 2008). Scale bars, 1µm.
1.2 Apoptosis
1.2.1 Introduction
Apoptosis is the most studied and best understood form of PCD. This process exists
in all multicellular organisms. Interestingly, the absence of this cellular process compared to
its importance causes relatively minor malformations in the development of C. elegans,
compared to higher organisms such as mouse or human. Apoptosis can be triggered through
two major pathways: 1) the extrinsic (death-receptor) pathway and 2) the intrinsic
(mitochondrial) pathway. However, some organisms including the nematode C. elegans
possess only the intrinsic pathway.
1.2.2 The mechanism of apoptosis in C. elegans
The core apoptotic machinery was first described in C. elegans. This is a free-living
nematode, which is about 1 mm in length and lives in temperate soil environments, where it
13
feeds with bacteria. The transparent body renders C. elegans as a tractable model system for
developmental studies at the single-cell level. This transparency facilitates the study of
cellular differentation and other developmental processes like apoptosis in the intact
organism (Fig. 2). The cell number of the worm is fixed: adult hermaphrodites consist of 959
somatic cells and adult males possess 1031 somatic cells. The developmental fate of every
single somatic cell has been mapped, which provides the invariant cell lineage of the
nematode (Sulston and Horvitz, 1977, Sulston et al., 1983). Thus, the pattern of somatic cells
dying by apoptosis during development is highly reproducible. Moreover, the developmental
timing of these apoptotic events is essentially invariant (Ellis and Horvitz, 1986). In the
hermaphrodite, 131 out of the total 1090 somatic cells die during development, 113 in the
embryonic and 18 in the postembryonic phase (Sulston and Horvitz, 1977, Sulston et al.,
1983).
Figure 2. Apoptotic cell corpses in a comma stage
embryo (indicated by arrows) - Cells that
underwent apoptosis exhibit a raised-button-like
appearance in C. elegans analysed by Nomarski
optics.
The mechanism of apoptosis is divided into three steps:
-initiation and execution
-DNA fragmentation
-cell corpse engulfment
1.2.2.1 Initiation and execution
In loss of function mutants for egl-1, ced-4, or ced-3, cell death is blocked and all of
the 131 cells that normally undergo apoptosis remain alive (Ellis and Horvitz, 1986). Despite
the presence of extra cells, development in these cell death-defective mutant animals
proceeds normally, and their lifespan, behavior and general appearance are similar to wildtype worms. In contrast, loss of ced-9 function causes widespread ectopic cell death and,
thereby resulting in lethality (Hengartner et al., 1992). These genes encode pro- and anti14
apoptotic factors of the core apoptotic machinery. ced-9 normally inhibits, while egl-1, ced-4
and ced-3 promote apoptosis. Epistasis analysis using null mutants of egl-1, ced-9, ced-4 and
ced-3 resulted in description of a simple genetic pathway ordering the above genes. egl-1
acts upstream of ced-9 to induce cell death, ced-9 acts upstream of ced-4 to inhibit cell
death, and ced-4 acts upstream of ced-3 to promote cell death (Conradt and Horvitz, 1998;
Hengartner et al., 1992; Shaham and Horvitz, 1996) (Fig. 6).
Biochemical and cell biological analyses show that CED-4 physically interacts with
CED-9 in vitro (Chinnaiyan et al., 1997; Spector et al., 1997; Wu et al., 1997). Furthermore,
endogenous CED-9 and CED-4 proteins co-localize at the mitochondrial surface, and the
mitochondrial localization of CED-4 is dependent on CED-9 activity (Chen et al., 2000). CED-4
has been shown to interact with CED-3 in vitro (Chinnaiyan et al., 1997; Yang et al., 1998).
Ectopic egl-1 expression in C. elegans embryos results in the translocation of CED-4 to
perinuclear membranes and ectopic programmed cell death (Chen et al., 2000). CED-4
translocation from mitochondria to perinuclear membranes appears to be initiated by the
binding of EGL-1 to CED-9, which induces conformational change in the CED-9 protein (Yan
et al., 2004). This leads to the release of CED-4 from the CED-9/CED-4 complex (Conradt and
Horvitz, 1998; del Peso et al., 1998; Parrish et al., 2000) (Fig. 3).
EGL-1-induced CED-4 translocation is important for the activation of CED-3 and,
hence, programmed cell death (Chen et al., 2000). CED-4 translocation promotes CED-4 selfoligomerization, which is thought to bring CED-3 proenzymes to close proximity for selfactivation (Yang et al., 1998) (Fig. 3). The C. elegans genome contains three additional
caspase-like genes, but it is currently unclear if any of these genes is involved in programmed
cell death (Shaham, 1998).
Figure 3. The core molecular machinery of apoptosis in C. elegans - EGL-1 binds to CED-9, which
leads to the dissociation of CED-4 from the CED-9 complex. After dimerization, CED-4 activates CED-3
,resulting in the initiation of apoptosis.
15
1.2.2.2 DNA fragmentation
Using DNA-staining techniques such as TUNEL-staining, ten genes have been
identified to be involved in nuclear DNA degradation during apoptosis (Parrish et al., 2001;
Parrish and Xue, 2003; Wang et al., 2002; Wu et al., 2000). These include nuc-1 (nuclease
defective), cps-6 (CED-3 protease suppressors), wah-1 (worm AIF homolog), crn-1 to crn-6
(cell deathrelated nucleases), and cyp-13 (cyclophilins). Genetic analysis indicates that these
genes act in multiple pathways and at different stages to promote DNA degradation and
apoptosis. cps-6, wah-1, crn-1, crn-4, crn-5 and cyp-13 constitute one pathway and crn-2 and
crn-3 participate in another one (Parrish and Xue, 2003; Wang et al., 2002).
Mutational inactivation of any of these genes results in the accumulation of TUNELpositive cells in C. elegans embryos. This suggests that these genes are important in the
elimination of TUNEL-reactive DNA breaks generated during apoptosis (Parrish et al., 2001;
Parrish and Xue, 2003). In addition, lack of activity of DNA fragmentation genes delayed
appearance of embryonic cell corpses during development indicating that nuclear DNA
degradation is important for normal progression of the apoptotic process. Most of these
genes can even promote cell suicide since deficiency in their activity, in a sensitized genetic
background, caused reduced number of cell death. Inactivation of DNA degradation
pathways causes also a synthetic defect in cell corpse engulfment. This implies that DNA
degradation process and cell corpse removal may tightly connected (Parrish and Xue, 2003).
1.2.2.3 Cell corpse engulfment
Unlike in higher organisms, in the soma of C. elegans there are no specialized cell
types for corpse engulfment, rather it is performed by the neighbouring cells. Cells, dying by
apoptosis, expose the "eat-me" signals on their surface, which are recognized by the
engulfing cells (Fadok et al., 2001). These signals are then transduced to the cellular
machinery in the engulfing cell to trigger the phagocytic process (Hedgecock et al., 1983).
Genetic analysis has identified eight genes participating in this process. They function in two
parallel pathways to promote cell corpse engulfment: ced-1, ced-6 and ced-7 functioning in
one pathway and psr-1, ced-2, ced-5, ced-10 and ced-12 in the other pathway (Chung et al.,
2000; Ellis et al., 1991; Gumienny et al., 2001; Wang et al., 2003; Wu et al., 2001; Zhou et al.,
2001). ced-7 activity is required in both the dying cell and the engulfing cell. All the other
16
genes from this list act only in the engulfing cell to promote corpse removal (Reddien and
Horvitz, 2004; Wu and Horvitz, 1998).
ced-1, ced-6 and ced-7 encode components of a signaling pathway involved in cellcorpse recognition. CED-1 may function as a phagocytic receptor recognizing corpses, since
CED-1 protein was found to cluster around cell corpses (Zhou et al., 2001). CED-7 may play a
role in promoting cell-corpse recognition by CED-1 (Wu and Horvitz, 1998; Zhou et al., 2001).
The CED-6 protein directly binds to the intracellular domain of CED-1 (Su et al., 2002) and
acts as a signaling adaptor (Fig. 4).
Figure 4. Molecular model for the process of cell corpse engulfment - In the CED-1, CED-6, and CED7 mediated pathway, CED-1 and CED-7 act on the surface of the engulfing cell and recognize signal(s)
from the surface of the dying cell. After transducing signal to CED-6, the phagocytotic machinery is
activated. CED-7 acts in the dying cell, too. The other pathway involves PSR-1, CED-2, CED-5, CED-10
and CED-12. PSR-1 acts in the engulfing cell to recognize PS. The signal is transduced through the
CED-2/CED-5/CED-12 complex to activate CED-10. There should be other engulfment receptors that
also act in the same pathway.
ced-2, ced-5, ced-10 and ced-12 encode conserved components of the Rac GTPase
signaling pathway. These genes are involved in the regulation of actin cytoskeleton
rearrangement, which is essential for cell migration and corpse engulfment. The
phosphatidylserine receptor-like protein PSR-1 specifically recognizes apoptotic cells that
present the engulfment-inducing signal phosphatidylserine (PS) (Fadok et al., 2001), and
17
promote the formation of the CED-2/CED-5/CED-12 complex. Genetic evidence indicates
that ced-2, ced-5 and ced-12 function at the same step, but upstream of ced-10. The GTPase
CED-10 is activated by binding to CED-5 and CED-12 (Fig. 4). CED-10 is the C. elegans
homolog of mammalian Rac GTPase proteins (Reddien and Horvitz, 2000), which control
cytoskeletal dynamics. In addition to CED-10, two other Rac-like GTPases, MIG-2 and RAC-2,
also participate in phagocytosis (Lundquist et al., 2001).
1.2.3 The mechanism of apoptosis in mammals
The mechanism of apoptosis in vertebrates is much more complex than it was
described in C. elegans. Homologs from mammals have been identified for each of the C.
elegans core apoptotic genes: CED-3 is a caspase-9 like protein; CED-4 is a homolog of the
adaptor protein apoptosis activating factor 1 (Apaf-1), which promotes assembly and
activation of caspases; CED-9 is a multidomain Bcl-2 family member; and EGL-1 is similar to
pro-apoptotic BH3-only proteins (Hengartner, 2000).
A central step in the process of apoptosis is the activation of caspases. They
constitute a family of cysteine proteases that are expressed in almost every cell as inactive
precursors (zymogens) with little or no protease activity (Hengartner, 2000; Thornberry and
Lazebnik, 1998). In response to death-inducing stimuli, specific aspartic residues of caspases
are cleaved, which results in the removal of an inhibitory N terminal domain and production
of a large and a small subunit, thereby activating the enzyme. The active protease is formed
by heterotetramers of the subunits, leading to the demolition phase of apoptosis
(Hengartner, 2000). However, not all mammalian caspases participate in apoptosis. Some of
them are activated during innate immunity and function to regulate cytokine processing and
maturation (Martinon and Tschopp, 2004).
The caspase family has been traditionally subdivided into initiator and effector
caspases (Hengartner, 2000; Thornberry and Lazebnik, 1998). Effector caspases have short
prodomains and are thought to accomplish apoptosis after proteolytic processing performed
by initiator caspases. Initiator caspases have long prodomains that bind large adaptor
molecules to promote multimerization and caspase activation. The prodomain of
mammalian caspase-9 associates with the adaptor protein Apaf-1 and cytochrome c to form
the apoptosome complex, and initiates apoptosis (Rodriguez and Lazebnik, 1999).
18
Figure 5. The molecular mechanism of apoptosome assembly - The anti-apoptotic Bcl-2 inhibits the
pro-apoptotic Bax. When one of the BH3-only family members (e.g. Bid or Bad) blocks Bcl-2, Bax form
oligomers, which results in MOMP, thus release of Cytochrome c. This leads to apoptosome
formation, therefore activation of caspase-3 and apoptosis. (based on van Empel and de Windt,
2004)
For apoptosome assembly, release of cytochrome c from the mitochondria is a
crucial step which is regulated by members of the B cell lymphoma 2 (Bcl-2) protein family
(Youle and Strasser, 2008). This family can be divided into three subfamilies depending on
the number of Bcl-2 homolog (BH) domains. The anti-apoptotic subfamily members possess
a BH4 domain (BCl-2). The two other subfamilies are pro-apoptotic, they usually lack the BH4
domain (BAX, BAK, and BOK) or solely display BH3 (BH3-only proteins, e.g. BAD and BID).
BH3-only family members antagonize the anti-apoptotic effect of Bcl-2, thereby enabling the
oligomerization of BAK/BAX within the mitochondrial outer membrane (MOM). This
contributes to the mitochondrial outer membrane permeabilization (MOMP) and allows the
release of cytochrome c and other proteins from the intermembrane space into the cytosol,
which results in the formation of the apoptosome (Fig. 5) (Martinou and Youle, 2011). The
apoptosome then promotes the activation of executor caspases such as caspase-3 and -7 by
their cleavage, which destroys the cell by cleaving functional proteins (Hengartner, 2000).
19
Caspase activation can also occur via association with death receptors, such as CD95, leading
to the oligomerization and activation of an initiator caspase, caspase-8 (Strasser et al., 2009).
This is called the extrinsic (death-receptor) pathway of apoptosis.
During apoptosis in mammalian cells, the DNA fragmentation phase is mediated by
several nucleases. The process of engulfment is accomplished by a group of specialised cells
called phagocytes. Dying apoptotic cells secrete ‘‘find me’’ and ‘‘eat me’’ signals to attract
and recruit phagocytes. The first ‘‘find me’’ signal to be identified is lysophospatidylcholine
(LPC). Additional molecules may act as ‘‘find me’’ signal, such as sphingosine 1 phosphate
(S1P) and chemokine ligand 1 (CXCL1) (Gude et al., 2008; Truman et al., 2008). LPC is
released by apoptotic cells in a caspase-3-dependent manner (Lauber et al., 2003).
The best-studied ‘‘eat me’’ signal is phosphatidylserine (PS), a component only
exposed to the extracellular surface during apoptosis (Fadok et al., 1992). This process is also
caspase dependent (Martin et al., 1996), but the exact mechanism in not yet known. PS can
be bound directly by a phagocyte receptor or indirectly by an adaptor molecule that
recognizes it, thus mediating the interaction with the phagocyte receptor (Nagata et al.,
2010).
Apoptotic cell engulfment is mediated by the Rho family of GTPases such as Rac1,
RhoA, and Rab5 (Nakaya et al., 2006). During ingestion, the apoptotic cell is converted into
its basic building blocks: amino acids, nucleotides, fatty acids, and monosaccharides in the
lysosomes. Engulfment is not just the conclusion of apoptosis, but also plays a decisionmaking role in cell death (Li and Baker, 2007).
1.2.4 Regulation of Apoptosis
According to our knowledge, in C. elegans apoptosis is regulated mostly by cell
specific transcription factors. Hence, the activity of EGL-1 is regulated at the transcriptional
level. In the soma of C. elegans, the major apoptosis promoting factor is EGL-1, which is
expressed in cells destined to die (Conradt and Horvitz, 1999; Thellmann et al., 2003).
Depending on the cell type, different transcriptional regulators control the expression of egl1. For example, in the hermaphrodite-specific neurons (HSN), the transformer-1 (TRA-1)
transcription factor represses egl-1 expression in hermaphrodites, thus HSNs survive
(Conradt and Horvitz, 1999). However, in males the lack of TRA-1 activity results in egl-1
expression, which in turn leads to apoptosis of HSNs (Fig. 6). The neuro-secretory
20
motorneuron (NSM) cells differentiate into serotonergic neurons, while their sisters, the
NSM sister cells, undergo programmed cell death during embryogenesis. In these latter cells,
a heterodimer, composed of two basic helix-loop-helix (bHLH) proteins, HLH-2/HLH-3
(Krause et al., 1997), is at least partially required for the egl-1-dependent cell death. In these
cells, a factor called cell death specification-1 (CES-1) blocks egl-1 expression, thereby
promoting survival. In normal conditions the expression of ces-1 is repressed by the basic
leucine zipper (bZIP) transcription factor CES-2 (Metzstein and Horvitz, 1999; Thellmann et
al., 2003) (Fig. 6.). Hence, HLH-2/HLH-3, CES-1 and TRA-1 are direct regulators of egl-1
transcription. These observations suggest that the pattern of egl-1 expression is precisely
regulated by a cascade of positive and negative factors in C. elegans (Fig. 6).
Figure 6. Genetic pathway of programmed cell death in C. elegans - The regulation of apoptosis is
cell specific in C. elegans. The expression of egl-1 is regulated by TRA-1 in HSNs, and by HLH-2/3 and
CES-1 in the NSM sister cells. The core apoptotic machinery functions downstream of EGL-1. Finally in
the "execution" phase, two partially redundant pathways mediate the engulfment of cell corpses and
the fragmentation of chromosomal DNA. Arrows represent activation, bars indicate inhibition.
(Conradt and Xue, 2005)
Evidences exist about the regulation of apoptosis at the level of proteins as well.
Inhibitor of cell death-1 (icd-1) has been reported to inhibit apoptosis in cells that are
normally fated to live (Bloss et al., 2003). The apoptotic function related to ICD-1 requires
CED-4, but not CED-3. However, the exact role of ICDs is unknown. It has also been shown
that caspase-2 (csp-2) and caspase-3 (csp-3) block apoptosis in the germ cells and in the
soma, respectively (Fig. 7) (Geng et al., 2009). Although homologs of the mammalian
inhibitor of apoptosis (IAP) protein group have not been identified in C. elegans, partial
caspase homologs, the CSPs, were found to protect cells from apoptosis as functional
orthologs of IAPs.
In higher organisms, a complex regulatory network is present in the cytoplasm,
which regulates apoptosis. Procaspases are widely expressed in living cells and have a low,
21
but significant protease activity, thus the negative regulation of caspases is very important.
IAP proteins belong to the family of caspase inhibitors. They bind to and block caspases
through their baculovirus inhibitory repeat (BIR) domain (Vaux and Silke, 2005). After the
discovery of IAPs in insect viruses, where they prevent apoptosis of infected cells (Crook et
al., 1993), a family of related proteins has been described in both Drosophila and mammals
(Vaux and Silke, 2005). The Drosophila IAP family member Diap1 functions as an E3 ubiquitin
ligase, targeting caspases in living cells, and promoting self-conjugation and degradation in
apoptotic cells (Ryoo et al., 2002; Ryoo et al., 2004). The best-studied mammalian IAP is the
X-linked inhibitor of apoptosis (XIAP) protein (Eckelman and Salvesen, 2006). Similar to
DIAP1, XIAP also functions as an E3 ubiquitin ligase to inhibit caspases in vivo (Schile et al.,
2008). However, compared to Drosophila, inactivation of XIAP causes only relatively ‘‘mild’’
phenotypes, because of the redundancy among mammalian IAPs (Schile et al., 2008).
In cells destined to apoptosis, IAPs are inactivated by specific antagonists originally
discovered in Drosophila (Kornbluth and White, 2005). Deletion of three closely linked genes
-reaper, hid, and grim (RHG)- preclude all apoptotic cell death in Drosophila (Fig. 7) (White et
al., 1994). On the other hand, ectopic expression of these genes can lead to induction of
apoptosis (White et al., 1996). The protein products of RHG genes contain a short N terminal
peptide motif called IAP-binding motif (IBM). The IBM is structurally conserved between
Drosophila and mammalian IAP antagonists, and is required for the inhibition of IAPs (Shi,
2002).
The RHG genes are transcriptionally induced by multiple death-inducing signals such
as developmental signals mediated by segmentation genes, steroid hormones, Dpp, Notch
signaling, JNK and various forms of cellular stress or injury (Steller, 2008). The promoters of
these genes share common cis-regulatory modules containing binding sites for HOX
transcription factors, nuclear hormone receptors, AP-1, and p53 (Brodsky et al., 2000; Jiang
et al., 2000; Christich et al., 2002; Lohmann et al., 2002; Zhang et al., 2008; Tan et al., 2011).
They also have a common epigenetic regulation mediated by Polycomb group proteins and
histon-modifying enzymes.
22
Figure 7. The core apoptotic machinery and its regulators in different organisms - The apoptotic
gene cascade is highly conserved, most of the core apoptotic genes have their orthologs in almost all
animal phyla (same colors indicate orthologous proteins). Also, the direct regulator proteins have
their „real” or at least fuctional orthologs. Arrows represent activation, bars indicate inhibition.
Mammalian IAP antagonists containing an IBM have also been discovered, (e.g.,
Smac/Diablo and HtrA2/Omi) (Fig. 7) (Verhagen et al., 2000; Suzuki et al., 2001). In contrast
to RHG proteins, these are localized within the mitochondrial intermembrane space, and
require MOMP for their release and binding to cytosolic IAPs (Green and Kroemer, 2004).
Another mammalian IAP antagonist is ARTS (apoptosis-related protein in the TGF-β signalling
pathway) (Larisch et al., 2000; Gottfried et al., 2004). Although ARTS does not contain a
recognizable IBM, it is localized, like RHG proteins, to the MOM, and it is a specific inhibitor
of XIAP (Gottfried et al., 2004; Garcia-Fernandez et al., 2010; Edison et al., 2011).
In summary, the activation of caspases is under dual control by both activators and
inhibitors, which in turn are subjects to many layers of regulation. The complexity of caspase
regulation network augments as organismal complexity rises. In higher species additional
backup mechanisms are necessary to prevent the survival of unwanted cells.
23
1.2.5 The functions of apoptosis
In most animal species, the function of apoptosis is necessary not only during
different phases of development but also during adulthood. Four major roles are ascribed to
apoptosis: 1) drive morphogenesis, 2) deletion of unnecessary tissues or complete organs, 3)
regulation of cell number, and 3) elimination of certain cells.
1.2.5.1 Morphogenesis
Apoptosis is essential for organogenesis and tissue remodeling. The best-known
example may be the formation of digits in higher vertebrates where elimination of the
interdigital webs is driven by PCD (Lindsten et al., 2000). Although apoptosis is the major cell
death mechanism in developing limbs, inactivation of pro-apoptotic genes in mouse only
partially prevents the removal of the interdigital tissue. This suggests that redundant
mechanisms exist when apoptosis is absent (Yuan and Kroemer, 2010). In Drosophila,
apoptosis is crucial in leg joint formation and in the morphogenesis of segments, mostly in
the developing head (Lohmann et al., 2002). In the male fly, apoptosis is also required for
permitting tissue rotation that drives looping morphogenesis of the genitalia (Kuranaga et
al., 2011). In chicks apoptosis participates in sculpting the future inner ear (Avallone et al.,
2002). It plays a fundamental role in the generation of the four-chamber architecture of the
heart (Abdelwahid et al., 2002).
1.2.5.2 Deletion of structures
During development, several structures that serve a transient function, for example
evolutionary relics, structures that are required in only one sex, or structures that are
temporarily necessary, are removed by apoptosis. In insects, most of the larval tissues are
partially eliminated by apoptosis (Baehrecke, 2002). During metamorphosis juvenile
structures are removed. For example, in amphibians the tail and intestine of the tadpole are
deleted by apoptosis. In fish and amphibians, pronerphric tubules form functioning kidneys,
which do not function in mammals, thus they are eliminated during mammalian
embryogenesis. In female mammals, oviducts and uterus is formed by the Müllerian duct,
which is deleted in males. Also in males, the Wolffian duct forms the vas deferens,
epididymis, and seminal vesicle, but it is eliminated in females by apoptosis (Jacobson et al.,
1997).
24
1.2.5.3 Regulation of cell number
In developing tissues and organs there is a fine balance between cell division and
apoptosis to achieve the adequate cell number. For example in the nervous, immune, or
reproductive system, cells are overproduced and superfluous ones are removed by
apoptosis. In human female embryos nearly 80% of the oocytes is eliminated by PCD, and in
almost all cases, these dying cells show apoptotic features (Reynaud and Driancourt, 2000).
Furthermore, more than half of all neurons generated in the mammalian central nervous
system are eliminated by apoptosis (Barres and Raff, 1999). Competition for limiting
amounts of survival signals is responsible for the appropriate numbers of different cell types
in a tissue. This strategy was first observed in Drosophila (Bergmann et al., 1998). Cells that
divide at different rates also compete with each other. In this case, slower proliferating cells
are eliminated from the population by overgrowing of more rapidly dividing cells. Cell
competition may contribute to tissue homeostasis and selection of the ‘‘fittest’’ cells
(Moreno, 2008). This phenomenon is also seen in mammals (Moreno, 2008; Bondar and
Medzhitov, 2010). All of these observations suggest fine tuned interplay among the
processes of cell division, differentiation and death.
1.2.5.4 Elimination of unnecessary and dangerous cells
Apoptosis also protects animal tissues by deleting cells that are abnormal and
potentially dangerous during animal development and adult life. For example in the human
immune system B and T lymphocytes have to pass a strict selection, demonstrating a
functional antigen receptor that is not autoreactive in order to evade cell death (Opferman
and Korsmeyer, 2003). If a cell is proved to be self-reactive, which could lead to
autoimmunity, it is eliminated by apoptosis. Viral infection, unrepaired DNA damage, cell
cycle perturbations, cell fate and differentiation defects could also lead to the deletion of
cells via apoptosis (Abrams et al., 1993; Rossel and Capecchi, 1999; Derry et al., 2001,
Vousden and Prives, 2009; Malumbres and Barbacid, 2009; Koto et al., 2011). In all of these
cases, apoptotic cell death serves as a ‘‘quality control’’ mechanism.
25
1.3 Autophagy
1.3.1 Introduction
Autophagy is a highly regulated self-degradation process of all eukaryotic cells. Its
mechanism is evolutionarily conserved from yeast to mammals. The basal, constitutive level
of autophagy plays an important role in cellular homeostasis through the elimination of
damaged/old organelles, as well as the turnover of long-lived proteins and protein
aggregates. Thus it maintains quality control of essential cellular components. Furthermore,
when cells are under environmental stress, such as nutrient starvation, hypoxia, oxidative
stress, pathogen infection or irradiation, the level of autophagy can be dramatically elevated
as a cytoprotective response, resulting in adaptation and survival. On the other hand,
excessive autophagy can lead to cell death under certain circumstances.
There are three major types of autophagy in eukaryotic cells: macroautophagy,
microautophagy and chaperone-mediated autophagy (CMA), all of which converge on
lysosomal degradation, but are mechanistically different from each other (Fig. 8). During
macroautophagy, parts of the cytoplasm, containing macromolecules or cellular organelles
(in particular, mitochondria), are sequestered into a double-membrane vesicle, termed
autophagosome. Autophagosome is then fused with a lysosome, thereby forming a structure
called autolysosome, where the separated cytoplasmic content is degraded by lysosomal
hydrolases. The resulting end-products of autophagic breakdown are transported back into
the cytosol through membrane permeases for synthetic processes (Wang and Klionsky,
2003). Microautophagy means the direct engulfment (i.e., invagination) of the cytoplasm at
the lysosome surface (Mijaljica et al., 2011). In the process of CMA, the unfolded, soluble
proteins carrying the KFERQ pentapeptid motif are translocated by chaperons to the
membrane of the lysosome, and are transported directly into the lysosome lumen via the
LAMP-2A transmembrane protein (Li et al., 2011).
26
Figure 8. The three well-known types of autophagy are the chaperon-mediated- (CMA), microautophagy and macroautophagy - Specific proteins that contain the KFERQ pentapeptid motif can be
degraded by CMA. By a process called microautophagy, the lysosome can directly engulf and degrade
a part of the cytoplasm. During macroautophagy, an isolating double-membrane separates a part of
the cytoplasm. The enclosed vesicle, termed autophagosome, then fuses with a lysosome and the
newly formed autolysosome digests the sequestered cytoplasmic content.
Macroautophagy (hereafter referred to as autophagy) is the most important type of
autophagy. It plays important physiological roles in human health and disease. Dysfunction
of this process in humans can cause pathologies such as cancer, neurodegeneration and
pathogen infection.
1.3.2 The mechanism of autophagy
Although autophagy was first identified in mammalian cells more than five decades
ago, the molecular understanding of this process only started in the second part of the ’90s.
The most important step of this was the discovery of autophagy-related (Atg) genes in the
yeast Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993; Klionsky et al., 2003), and then
the identification of their homologs in higher eukaryotes such as mouse and human (Table
1.). Atg proteins that are essential for autophagosome formation are called the ‘core’
molecular machinery.
Table 1. The core autophagy genes and their orthologs in S. cerevisiae, C. elegans and Homo
sapiens - The autophagy gene cascade was first described in yeast which consists of the AuTophaGy
27
related genes (ATG). Later, orthologs were identified in higher eukaryotes as well, although there are
differences in the cascade from taxon to taxon.
yeast
nematode
human
ATG1
unc-51
ULK1, ULK2
ATG2
atg-2
ATG2A and B
ATG3
atg-3
ATG3
ATG4
atg-4.1, atg-4.2
ATG4A, B, C and D
ATG5
atg-5
ATG5
ATG6
bec-1
BECN1
ATG7
atg-7
ATG7
ATG8
lgg-1, lgg-2
GABARAP,
MAP1LC3A and B
ATG9
atg-9
ATG9A and B
ATG10
atg-10
ATG10
ATG11
atg-11
-
ATG12
lgg-3
ATG12
ATG13
atg-13
-
function
protein serin/threonin kinase,
participates in induction of
autophagy
membrane protein,participates in
Atg9 recyclisation
E2-like enzyme, necessary for the
Atg8-PE conjugate formation
cystein protease, required for
processing Atg8
member of the Atg12-Atg5 complex
coiled-coil protein, member of the
class III phosphatidylinositol 3kinase complex
E1 ubiquitin-activating-like enzyme,
necessary for both conjugation
systems
membrane protein, component of
the Atg8-PE conjugating system
transmembrane protein, involved in
membrane delivery to the PAS
E2-like enzyme, necessary for the
formation of Atg12-Atg5 complex
coiled-coil domain protein, required
for recruiting other proteins to the
(PAS)
hydrophilic protein, member of the
Atg12-Atg5 complex
member of the Atg1 complex
coiled-coil protein, subunit of
phosphatidylinositol 3-kinase
complex I
ATG14
epg-8
-
ATG16
atg-16.1, atg16.2, atg-16.3
ATG16L1 and L2
ATG17
-
FIP200
ATG18
atg-18
WIPI, WIPI2
ATG19
-
-
28
interacts with Atg12p-Atg5p
conjugates to form Atg12p-Atg5pAtg16p multimers
coiled-coil protein, member of the
Atg1 complex
participates in Atg9 recyclisation
cargo-receptor protein, involved in
the cytoplasm to vacuole transport
coiled-coil protein, involved in the
cytoplasm to vacuole transport
ATG20
-
-
ATG21
-
WDR45L
ATG22
-
-
ATG23
-
-
ATG24
-
-
ATG26
-
-
ATG27
-
-
ATG29
-
-
ATG31
-
-
ATG32
-
-
ATG33
-
-
ATG34
-
-
-
Y69A2AR.7
Atg101
VPS34
let-512
HVPS34 and PIK3C3
member of the Class III
phosphatidylinositol 3-kinase
complex
VPS15
ZK930.1
-
member of the Class III
phosphatidylinositol 3-kinase
complex
-
epg-2
-
-
epg-3
VMP1
-
epg-4
epg-5
EI24/PIG8
KIAA1632
-
epg-6
WDR45
WD repeat containing protein,
involved in binding of
phosphatidylethanolamine to ATG8
transmembrane protein, required
for lysis of autophagic vesicles
coiled-coil protein, involved in the
cytoplasm to vacuole transport
phosphatidylinositol-3-phosphate
binding protein, involved in the
cytoplasm to vacuole transport
glycosyltransferase, involved in the
biosynthesis of sterol glucoside
transmembrane protein, required
for cycling of ATG9
interacts with Atg17 and localizes
to the PAS
form a complex with Atg17 and
Atg29 that localizes other proteins
to the PAS
29
MOM protein, required to initiate
clearence of mitochondria by
autophagy (mitophagy)
mitophagy-specific protein
receptor protein, involved in
selective autophagy
interact with Atg13
multiple coiled-coil domain protein,
regulates the association of P
granules with autophagic structures
regulates the early step of
autophagosome formation
transmembrane protein
regulates the final degradation step
WD40 Repeat PtdIns(3)P-Binding
Protein
The mechanism of autophagy can be divided into four steps:
-induction
-vesicle nucleation
-vesicle elongation and closing
-maturation of autophagosome and fusion with the lysosome
1.3.2.1 Induction
The induction of autophagy is mediated by the Atg1 complex in yeast. The Atg1
complex is required for recruitment of other Atg proteins to the phagophore assembly site
(pre-autophagosomal structure or PAS) and also regulates the movement of Atg proteins
between PAS and other membranes (Cheong et al., 2005, Cheong et al., 2008). PAS is the
initiation site, where most of the core Atg proteins are recruited for autophagosome
formation. Atg1 is a serine/threonine kinase that forms a complex with Atg13 and Atg17.
The nutrient sensor target of rapamycin complex 1 (TORC1) is a major negativ regulator of
autophagy. During starvation TORC1 inactivation leads to dephosphorylation of Atg13, thus
an increased Atg1–Atg13–Atg17 complex formation and autophagic activity (Fig. 9) (Kamada
et al., 2000).
In mammals a whole family of Atg1 proteins has been described, but only a fraction
of them modulates autophagy. The UNC-51-like kinases 1 (ULK1) and 2 (ULK2) show the
highest similarity to yeast Atg1 and appear to be closely related. It has been demonstrated
that silencing of ULK1 or ULK2 by siRNA inhibits autophagy in HEK293 cells (Jung et al.,
2009). ULK1 and ULK2 also participate in the complex that includes the mammalian homolog
of Atg13 (mAtg13) and the scaffold protein FIP200 (an ortholog of yeast Atg17). Both
mAtg13 and FIP200 are essential for autophagy (Ganley et al., 2009). Independently of its
phosphorylation state, mAtg13 directly interacts with ULK1, ULK2 and FIP200. FIP200 also
binds to ULK1 and ULK2 independently of the nutrient status, in contrast to the yeast Atg1–
Atg17 interaction (Hosokawa et al., 2009). Under nutrient-rich conditions, mammalian
TORC1 (mTORC1) is also involved in the formation of the large ULK1–Atg13–FIP200 complex,
and in turn after nutrient restriction mTORC1 dissociates from the ULK1 complex, resulting
in the activation of autophagy (Fig. 9) (Hosokawa et al., 2009). There are several
phosphorylation events between these proteins, but their exact functions are not yet
known.
30
Figure 9. The activation of Atg1 complex in yeast and mammals - Under starvation or rapamycin
treatment, the yeast TORC1 is inhibited, thus it cannot phosphorylate Atg13. Dephosphorylated
Atg13, together with Atg1 and Atg17, can form an active Atg1 complex, therefore iniciates
autophagy. In mammals, TORC1 is directly involved in the formation of the inactive Atg1 complex
during normal environmental conditions. Under starvation or rapamycin treatment, TORC1
dissociates, thus ULK1/2 and Atg13 are dephosphorylated, which leads to the activation of the
complex via a series of phosphorylation events. Arrows represent activation, bars indicate inhibition,
P means phosphorylation and crossed out P implies dephosphorylation.
1.3.2.2 Vesicle nucleation
The next step in the autophagic process is nucleation, the event that leads to the
formation of the initial autophagic structure called the phagophore at PAS. It is still an open
question where the autophagic membrane comes from. Existing data show that the source
of this membrane can be the mitochondrial membrane, endoplasmic reticulum, Golgi
apparatus or endosomes, but some researcher hypothetize that this structure can form also
de novo from cytoplasmic precursors (Tooze and Yoshimori, 2010).
The nucleation is the assembly of the initial phagophore membrane and autophagy
proteins to PAS. This process requires the function of the class III phosphatidylinositol 3kinase (PtdIns3K) complex. There is only one PtdIns3K in yeast called Vps34, and it is
involved in two different complexes, complexes I and II. Complex I contains Vps34, Vps15,
Atg6 and Atg14, and complex II consists of Vps34, Vps15, Atg6, and Vps38 (Fig. 10), but only
complex I is involved in autophagy (Kihara et al., 2001). The lipid kinase activity of Vps34 is
essential for generating phosphatidylinositol (3)-phosphate (PtdIns(3)P) at the PAS, thus
other Atg proteins can be recruited (Fig. 10).
31
Figure 10. The different complexes of the class III phosphatidylinositol 3-kinase (Vps34) - Complex I
and complex II share Atg6, Vps34 and Vps15. Atg14 and Vps38 give specificity to the complex I and II
(a), respectively. In yeast, complex I is involved in autophagy by assembling Atg proteins to PAS (b).
In mammalian cells, the core of the class III PtdIns3K complex includes hVps34,
Beclin1 (homolog of Atg6) and p150 (homolog of Vps15). Orthologs of Atg14 and Vps38 have
also been identified, they are called Atg14-like proteins (Atg14L or Barkor) and ultraviolet
irradiation resistance-associated gene product (UVRAG), respectively (Itakura et al., 2008).
Unlike Vps38 in yeast, UVRAG seems to participate in the regulation of several
different steps of mammalian autophagy. UVRAG competes with Atg14L for binding to Beclin
1, in a complex only one of them can interact with the Beclin 1–hVps34–p150 molecules
(Liang et al., 2006). Additionally the recently identified Rubicon (RUN domain and cysteinerich domain containing, Beclin 1-interacting) protein associates with UVRAG–Beclin 1–
hVps34–p150 and this complex localizes to the late endosome/lysosome (Zhong el al., 2009).
Since Rubicon reduces hVps34 activity, it regulates autophagosome maturation negatively.
It has also been published that depletion of the transmembrane protein VMP1
blocks starvation-induced and rapamycin-induced autophagy, whereas overexpression of
VMP1 triggers autophagy through an interaction with Beclin 1 even under nutrient-rich
conditions in mammalian cells (Ropolo et al., 2007). The possible role of this transmembrane
protein in this process might be recruiting Beclin 1 and other components in the class III
PtdIns3K complex to the phagophore.
1.3.2.3 Vesicle elongation and closure
Vesicle nucleation is followed by the vesicle elongation phase in which the function
of the two, partially overlapping ubiquitin-like protein (Atg12 and Atg8/LC3) conjugation
systems are crucial both in yeast and in mammals. Mice defective for these complexes have
32
smaller autophagosomes than the wild type and have either open-ended or multilamellar
vesicles (Sou et al., 2008). These data indicate that the two conjugation systems participate
in the elongation and closure of the phagophore.
Atg12 together with Atg5, Atg7, Atg10 and Atg16L are necessary for the formation
of the Atg16L complex. First Atg12 is conjugated to Atg5 in a reaction that requires Atg7 and
Atg10 (E1 and E2-like enzymes, respectively) (Tanida et al., 1999, Shintani et al., 1999). Then
the Atg12–Atg5 conjugate binds noncovalently to Atg16L, and these form a big multimeric
complex called the Atg16L complex (Fig. 11) (Mizushima et al., 1999). Although the
significance of the Atg12-Atg5-Atg16 complex in autophagosome formation has been shown,
its molecular function is still not clearly understood. One identified function of the Atg12Atg5 conjugate is to promote the formation of the Atg8-phosphatidylethanolamine (PE)
conjugate and targeting of the Atg8-PE to the PAS (Mizushima et al., 2003).
Atg8 is the key component of the other conjugation system, which controls the size
of the autophagosome. It may result from its ability to determine membrane curvature. The
newly generated Atg8/LC3 is cleaved by Atg4 at the C terminus (Kirisako et al., 2000). This
cytosolic LC3-I has a glycine residue at its C terminus, which conjugates to PE in a reaction
that also requires Atg7 as E1 and the E2-like enzyme Atg3 (Fig. 11) (Yang and Klionsky, 2009).
The lipidated form of LC3, called LC3-II attaches to both surfaces of the phagophore
membrane, but it is finally removed from the outer membrane of the autophagosome,
before it fuses with a late endosome or a lysosome.
It has been shown that these two ubiquitination-like systems are closely connected.
The Atg16L complex is found to function as an E3-like enzyme, and it can determine the sites
of Atg8/LC3 lipidation on the surface of the phagophore (Hanada et al., 2007, Fujita et al.,
2008). Interestingly, the Atg8/LC3 conjugation machinery seems to be essential also for the
formation of the Atg16L complex (Sou et al., 2008).
It is known that the yeast Atg9 protein contributes to membrane delivery to the
forming autophagosome (He et al., 2006). Upon starvation or rapamycin treatment, the
mammalian transmembrane protein mAtg9, encoded by the ortholog of yeast ATG9,
translocates to peripheral sites and overlaps with GFP-LC3-positive autophagosomes (Young
et al., 2006). It is suggested that mAtg9 could have the same function as its yeast ortholog.
33
Figure 11. The two ubiquitin-like protein conjugation systems and the interplay between them First, Atg12 conjugates with Atg5. This reaction is supported by Atg7 and Atg10 as E1- and E2-like
enzymes, respectively. Finally, Atg16 binds to the Atg12-Atg5 conjugate, and this complex forms
oligomers on the surface of the autophagosome. In the other system, Atg4 cleaves the C terminus of
Atg8. Then it binds to PE in a reaction that requires Atg7 and Atg3 (E1 and E2-like enzymes,
respectively). The two systems communicate with each other at several points.
1.3.2.4 Maturation of autophagosome and fusion with lysosome
In the next step, the autophagosome can go through maturation by fusion with
early or late endosomes or with multivesicular bodies. It has been described that Rab
proteins are required for these fusions (Fader et al., 2008, Chua et al., 2011). Finally the
autophagosome fuses with the lysosome which is mostly dependent on the protein
complexes present on the double membrane like Atg12-Atg5-Atg16, LAMP2 receptor and
the small GTPase protein Rab7 (Jager et al., 2004, Tanaka et al., 2000). The union between
vacuole and lysosome happens when Atg8-PE molecules leave the outer surface of the
autophagosome. Many other proteins participate in this fusion, like the SNARE proteins
34
(Longatti and Tooze, 2009). The formation of the autolysosome is followed by the
degradation process that is mediated by different proteases like lysosomal cystein protease
or cathepsin in mammals.
1.3.3 The regulation of autophagy
Because of its widespread roles and functions numerous signaling pathways,
genetic and environmental factors regulate autophagy and they constitute a complex
network. Starvation and both extra- and intracellular signals, such as RAS, AMPK, insulin/IGF1, TOR, p53, Sirt1, Bcl-2 are involved in the modulation of autophagy. The regulation of the
autophagic process seems to be considerably conserved from yeast to mammals.
1.3.3.1 The main regulator of autophagy: the insulin/IGF-1-TSC1/2-TOR
network
The insulin/IGF-1 signaling pathway influences numerous cellular functions such as
growth, differentiation, glucose homeostasis, and nutrient sensing. Upon association with
insulin-like growth factor (IGF) or other insulin-like ligands, the tyrosine kinase insulin-like
growth factor receptor (IGFR) undergoes autophosphorylation and becomes activated,
leading to the stimulation of two key signal-transducing components: the small GTPase Ras
and class I PtdIns3K (Yang and Klionsky, 2010). Class I PtdIns3K catalyzes the production of
PtdIns(3)P at the plasma membrane, which increases membrane recruitment of both
PKB/Akt (protein kinase B) and its activator PDK1 (phosphoinositide-dependent protein
kinase 1), leading to the activation of PKB/Akt (Coffer et al., 1998). The downstream
components of this pathway are the FoxO transcription factor isoforms. After
phosphorilation by PKB/Akt FoxOs remain in the cytoplasm, therefore they cannot enter the
nucleus and activate their target genes. FoxO1 and FoxO3 directly activate LC3b, GabarapL1
and Atg12L autophagy genes in mammals (Fig. 12) (Sengupta et al., 2009; Mammucari et al.,
2007; Zhao et al., 2007).
The target of rapamycin (TOR) serves as a master regulator of autophagy. It is a
highly conserved serine/threonine protein kinase that acts as a central sensor of growth
factors, nutrient signals, and energy status. TOR exists in two distinct complexes, TORC1 and
TORC2, which are conserved from yeast to mammals, and TORC1 can directly regulate
autophagy (see 1.3.2.1. the induction of autophagy).
35
Figure 12. The regulation of autophagy via the insulin/IGF-1 - TSC1/2 - TOR signaling network Signals through IGFR regulate autophagy in several different manners. The insulin/IGF-1 pathway
blocks the activity of its downstream transcription factors, the FoxOs. FoxO1 and FoxO3 are direct
positive modulators of the transcriptional activity of some autophagy genes. Furthermore, FoxO1
activates autophagy through the inhibition of TORC1. Additionally, the insulin/IGF-1 pathway inhibits
autophagy through the TSC1/2 complex-Rheb-TORC1 pathway. PKB/Akt has a positive feedback via
TORC2, thus it also participates in the modulation of autophagy. AMPK activates autophagy through
direct and indirect inhibition of TORC1. Furthermore, Ras can both activate and inhibit autophagy
through the Raf-1–MEK1/2–ERK1/2 pathway and via Akt, respectively.
Beside transcriptional regulation of autophagy genes mediated by FoxOs, the
insulin/IGF-1 signaling pathway acts on autophagy in different ways but always through TOR.
First, FoxO1 particitapes in the degradation of Raptor and mTOR, resulting in the inactivation
of TORC1 (Wu et al., 2008). Second, PKB/Akt activates mTORC1 through inhibiting a
downstream protein complex, the tuberous sclerosis 1/2 complex (TSC1/TSC2) (Inoki et al.,
2002). The TSC1/TSC2 heterodimer, which is a stable complex, senses the upstream inputs
from various kinases, including PKB/Akt. Phosphorylation of TSC2 by PKB/Akt leads to the
disruption of its complex with TSC1, and results in mTOR activation. TSC1/TSC2 acts as the
36
GTPase activating protein for Rheb, a small GTP-binding protein that activates TORC1 in its
GTP-bound form (Fig. 12). Finally, the mTORC2 complex is also involved in the regulation of
autophagy. Full activation of PKB/Akt requires mTORC2, and inhibition of PKB/Akt caused by
mTORC2 depletion reduces the phosphorylation of FoxO3 transcription factor, therefore
activates it. FoxO3 stimulates autophagy in muscle cells independently of the activity of
mTORC1 (Fig. 12) (Sarbassov et al., 2005).
1.3.3.2 Other regulators of autophagy
The AMP-activated protein kinase (AMPK) is another sensor of cellular
bioenergetics, specifically in response to cellular energy status. During nutrient and energy
depletion, AMPK is activated by a decreased ATP/AMP ratio. Active AMPK leads to
phosphorylation and activation of TSC1/TSC2, thereby inhibiting mTORC1 activity.
Accordingly, phosphorylation of TSC1/TSC2 by AMPK and by PKB/Akt, has opposite effects
on mTORC1 activity, thus energy and growth factor signaling converge on mTORC1.
Recently, it has been reported that AMPK also regulates mTORC1 signaling through an
alternative mechanism, whereby AMPK directly phosphorylates Raptor, and this is important
for the inhibition of mTORC1 signaling by AMPK. Summarizing, AMPK serves as a positive
regulator of autophagy (Fig. 12) (Shaw, 2009).
The small GTPase, Ras has opposing roles in autophagy regulation: it inhibits
autophagy by activating the PtdIns3K–PKB/Akt, mTORC1 pathway, and at the same time it
may induce autophagy via the Raf-1–MEK1/2–ERK1/2 pathway (Fig. 12) (Yang and Klionsky,
2010).
The tumor suppressor protein p53, the ‘guardian of the cellular genome’, has dual
positive and negative regulatory roles in autophagy induction. Upon genotoxic- or oncogenic
stress, the activation of p53 induces autophagy (Crighton et al., 2006). At the same time, in
case of several stimuli including starvation or ER stress, proteasomal degradation of p53 is
induced to support autophagy induction, thus p53 serves as a negative regulator of
autophagy (Tasdemir et al., 2008; Morselli et al., 2008). Importantly, it is the cytoplasmic p53
that exerts its inhibitory function on autophagy, in contrast to the transcriptionally active
nuclear p53 that promotes autophagy.
In mammals, the Bcl-2 protein family plays a dual role in autophagy regulation. Antiapoptotic proteins, such as Bcl-2 inhibit autophagy, whereas pro-apoptotic BH3-only
37
proteins, such as Bad induce autophagy. The binding of Bcl-2 to Beclin 1 disrupts the
association of Beclin 1 with hVps34, decreases Beclin 1 associated hVps34 PtdIns3K activity
and thereby inhibits autophagy. Starvation-induced autophagy requires Sirt1, both in vitro
(Lee et al., 2008) and in vivo (Morselli et al., 2010). Activated Sirt1 induces autophagy via its
capacity to deacetylate acetyl lysine residues in other proteins. It has been shown that Sirt1
deacetylates Atg5, Atg7, LC3 (Lee et al., 2008), and the transcription factor FoxO3 (Kume et
al., 2010), which can stimulate the expression of autophagic genes. Thus Sirt1 controls
autophagy at multiple levels, including the modification of autophagy core proteins and
transcription factors which control the expression of autophagic genes.
1.3.4 Functions of autophagy in animal development
The process of autophagy is primarily a stress response mechanism but it has
several different functions including regulation of lifespan, maintaining cellular homeostasis,
protection against intracellular pathogens. In addition it plays a role in animal development,
tissue differentiation and PCD. In my work I only focus on the developmental functions of
autophagy.
Studies in lower and higher eukaryotes revealed an essential role of autophagy in
differentiation and development. In the yeast Saccharomyces cerevisiae, autophagy is
required for normal spore formation (Tsukada and Ohsumi, 1993). The amoeba
Dictyostelium discoideum exists in a vegetative life state in soil where it feeds on bacteria.
When food is limited, it differentiates by aggregation to form a fruiting body. This process
requires autophagic cell death to carry the spore-forming cells to the stalk, enabling
sporulation and dispersal. As a first signal, autophagy is induced by starvation and cAMP, and
as a second signal the differentiation factor DIF-1 is required to induce PCD (Otto et al., 2003
and 2004).
In Drosophila melanogaster, during the transition from larvae to adult, larval tissues
including the salivary glands and midgut are removed. Studies examining elimination of the
salivary gland observed features of both apoptosis and autophagy (Berry and Baehrecke,
2007). According to this, genetic experiments show that deletion of salivary glands is only
partially blocked by inhibition of the canonical caspase activation machinery, suggesting that
other processes are required for their complete removal. Indeed, when autophagy is blocked
by loss-of-function mutations in Atg genes, degradation of salivary glands is delayed.
38
Furthermore, when both caspase and autophagy inhibition are combined, a severe delay in
removal is observed showing greater inhibition of degradation than in absence of either
mechanism alone. These genetic studies suggest a model whereby developmental PCD of
the salivary glands requires both apoptosis and autophagy acting in parallel pathways.
Removal of the larval midgut during larval–pupal transition is only dependent on
autophagy (Denton et al., 2009). Although, mutations of the canonical apoptotic machinery
do not affect midgut removal, but in the dying midgut autophagy markers accumulate and
inhibition of autophagy by loss-of-function mutations in Atg2 and Atg18 or knockdown of
Atg1 or Atg18 by RNA interference severely delays their removal (Fig. 13).
Figure 13. Removal of the Drosophila larval midgut is mediated by autophagy - Morphology of
midguts display a significant delay in midgut degradation both in Atg2 and Atg18 mutant background,
compared to controls, as seen by the presence of less contracted gastric caeca (arrows) (Denton et
al., 2009).
In the Drosophila ovary, certain germline nurse cells, somatic follicle cells and
defective egg chambers are removed by starvation-induced cell death mediated by
autophagy (Hou et al., 2008; Barth et al., 2011). It is also known that autophagy positively
regulates development of the Drosophila larval neuromuscular junctions (Shen and
Ganetzky, 2009).
In mammalian cells, autophagy seems to be essential in many stages during animal
development. It occurs at low levels in unfertilized oocytes but it is massively induced within
the embryo 4 hours after fertilization (Fig. 14) (Tsukamoto et al., 2008). This induction of
39
autophagy is completely dependent on fertilization. In this case autophagy could have the
same role as in C. elegans embryos, i.e., the elimination of paternal miochondria.
Figure 14. Induction of autophagy after fertilization - Unfertilized (left) and fertilized (right) oocytes
from female mice. GFP–LC3-positive puncta indicate autophagosomes (Tsukamoto et al., 2008). Scale
bar, 10 μm.
During embryogenesis autophagy is temporarily suppressed from the late one-cell
to middle two-cell stages and then reactivated. Some autophagy components are maternally
loaded; for example, elimination of the maternal Atg5 protein results in embryonic lethality
at the four-cell to eight-cell stages (Tsukamoto et al., 2008). The precise role of autophagy
during this stage is not yet known. One hypothesis predicts that normal levels of autophagy
maybe necessary for the production of sufficient amino acids for protein synthesis, based on
the reduced rate of protein synthesis in autophagy defective embryos. However, autophagy
may also be required for the active elimination of unnecessary proteins and organelles that
are no longer needed in fertilized oocytes. Thus, autophagy may assist in reorganization of
the intercellular millieu. Although high levels of autophagy have not been reported during
later stages of embryogenesis in mice, studies show autophagic activity in certain tissues
such as the developing thymus. The next significant autophagic wave is observed during the
early neonatal period in mice. Autophagy is actively induced in all neonatal tissues except
the brain until one or two days after birth (Kuma et al., 2004). Throughout mammalian
embryogenesis, necessary nutrients are supplied through the placenta. At birth, this supply
is terminated and neonates face severe starvation that induces autophagy.
A long-standing question in developmental biology has been how the erythroblast
loses its organelles. During erythroid differentiation, the nuclei are released from the cell,
40
but the mechanism(s) by which other organelles are eliminated are poorly understood.
Several recent studies suggest that mitochondrial clearance in reticulocytes is at least
partially dependent on autophagy (Takano-Ohmuro et al., 2000). For example it has been
described that Atg7−/− erythrocytes accumulate selectively damaged mitochondria
(Mortensen et al., 2010).
The clearance of mitochondria represents a developmental process that contributes
to the survival of lymphocytes. Elimination of mitochondria is known to be mediated by
autophagy, since both CD4+ and CD8+ T cells from haematopoietic cell-specific Atg7knockout mice have a higher number of mitochondria (Mortensen et al., 2010).
Accumulation of damaged or aging mitochondria results in higher level of reactive oxigen
species (ROS) and finally cell death. Furthermore, T-cell specific Atg5- and Atg7-knockout
mice show a decline in the number of peripheral T cells, the accumulation of the T-cell
mitochondria and enhanced apoptosis in mature T cells (Pua et al., 2007; 2009). Another
recently identified role of autophagy is the elimination of autoreactive T cells in the thymus
(Nedjic et al., 2008). The survival of developing B-cells is also dependent on autophagy, but
this process is independent from the amount of mitochondria (Pua et al., 2009).
During embryogenesis adipocytes differentiate from preadipocytes, which emerge
from multipotent mesenchymal precursors. Both in vitro and in vivo studies have confirmed
that autophagy contributes to cellular remodelling during adipogenesis. Primary mouse
embryonic fibroblasts (MEFs) can be differentiated into adipocytes in the presence of
adipogenic factors, but this process is significantly delayed or suppressed in primary Atg5−/−
MEFs (Baerga et al., 2009). Newborn animals lacking Atg5 and Atg7 have decreased white
adipose tissue mass and the features of the adipocytes are characteristic of brown adipose
tissue (Singh et al., 2009).
1.3.5 Autophagy in C. elegans
Since both the mechanism and the regulation of autophagy are highly conserved
across animal phyla, C. elegans shows only minor differences in these processes compared
to mammals or S. cerevisiae. However, in this aspect nematodes stand closer to mammals.
Almost all core autophagy genes have already been found in C. elegans, although some of
them seemed to be not present in this organism, since they were not found previously by
bioinformatics analysis. P granules are specialized RNA and protein aggregates that regulate
41
germ cell fate, and their degradation in somatic cells is mediated by autophagy during
embryogenesis (Zhang et al., 2009). Recently published screens, based upon this
observation, however uncovered some genes, named epg genes (ectopic P granules) (Tian et
al., 2010). They encode proteins that are functional orthologs of known members of the
mammalian autophagic machinery (Table 1).
Autophagy is required for the elimination of paternal mitochondria as well, after the
sperm entered the oocyte cytoplasm upon fertilization. In autophagy-defective zygotes,
paternal mitochondria and their genome remain even in the first larval stage (Sato and Sato,
2011; Rawi et al., 2011). The mechanism of autophagy is also necessary for dauer formation,
an alternative non-feeding stage of nematode development that is a response to harsh
environmental conditions (Meléndez et al., 2003).
In C. elegans the direct regulation of autophagy is not as complex as in mammals.
The insulin/IGF-1-TOR pathway is the main modulator here as well. daf-16, the ortholog of
the mammalian FoxOs, directly represses the expression of the daf-15/Raptor gene that
encodes a subunit of the TOR complex, thus activates autophagy (Jia et al., 2004). let363/CeTOR encodes the ortholog of mTOR in C. elegans. Interestingly, in contrast to
mammals only one TOR complex exists in this organism. However, it is thought to be similar
to the mTORC1, but it does not seem to be sensitive to rapamycin. Inactivation of let-363 by
RNAi activates autophagy, thus under normal conditions CeTOR inhibits autophagy.
Orthologs of TSC1 and TSC2 have not yet been identified in C. elegans.
42
2 Aims
2.1 Investigating simultaneous inactivation of autophagy and
apoptosis in C. elegans
During apoptosis, upstream regulatory signals lead to the activation of the caspase
cascade which kills the unnecessary cells in the developing organism. Thus apoptosis is
basically a mechanism to eliminate cells. On the contrary, the most important function of
autophagy, a mechanism evolved for degrading damaged intracellular components, is to
protect the cell from death. In some cellular settings, autophagy rescues the cell from
undergoing apotosis. However, it is known that in other cases autophagy can also kill the cell
or participates in its elimination, thus it assists to apoptosis.
The exact role of these mechanisms during embryonic and postembryonic
development of C. elegans is still not known. Apoptosis-deficient single mutant nematodes
are viable, fertile, and their morphology and behaviour do not differ significantly from that
of the wild type. Single loss-of-function mutants for several autophagy genes (eg. unc51/Atg1 and atg-18) are also viable and fertile. According to these, the model organism
C. elegans provides a good opportunity to examine how apoptosis and autophagy are
related to each other during development. Do they act as two friends who support each
other or as enemies in a battle?
To answer these questions first we created double mutant animals, lacking the
function of both autophagy and apoptosis. Then we examined their phenotypes compared
to wild type and the corresponding single mutants. We planned to use several null alleles or
hypomorphic mutations for genes that are involved in different steps of the autophagic
process. We also planned to involve different apoptosis deficient strains into our
experiments, such as loss-of-function mutations of genes influencing caspase-dependent
and –independent apoptosis as well.
Furthermore we wished to examine the level of apoptosis in different autophagic
mutant backgrounds, and vica versa the rate of autophagy in animals lacking apoptosis.
Several methods exist to measure apoptosis in C. elegans. We intended to use at least two of
them: 1) counting the number of apoptotic corpses and 2) applying the widely used
apoptosis-specific TUNEL staining. To quantify the level of autophagic activity we wished to
analyse the expression level and pattern of the autophagic marker GFP::LGG-1. The LGG43
1/Atg8 protein is located on the surface of the autophagosomes and autolysosomes, thus it
is widely used for visualizing autophagy.
2.2 Common regulation of autophagy and apoptosis
Preliminary results indicate that during development autophagy and apoptosis are
induced together, thus common regulatory factors should exist. We planned to search for
transcription factors that may act as regulators of both processes. Since our knowledge
about transcriptional regulation of autophagy genes is poor, we tried to find conserved
transcription factor binding sites of known apoptosis regulators in the 5’ regulatory region of
autophagy genes using bioinformatic methods.
We wished to validate our in silico results applying an in vitro technique called
electrophoretic mobility shift assay or EMSA. We planned to express the candidate
transcription factors and intended to use them for gel retardation analysis in which marked
oligonucleotides contain the potential binding sites. Finally, we wanted to confirm these
results in an in vivo model. We planned to design reporter constructs that contain 1) the
analysed regulatory sequences and 2) their mutant counterparts lacking the potential
binding sites. Next we intended to generate transgenic worms using the previous constructs
and to analyse their expression.
44
3
Materials and methods
3.1 Maintenance of C. elegans strains
Worms were grown in termostates at 15-20 °C on ∅ 5.5 cm plastic Petri dishes
containing nematode growth medium (NGM) agar (3 g NaCl, 2.5 g bacto-peptone, 17 g agar,
1 ml of 5 mg/ml cholesterol and ddH2O to 1 l are mixed, autoclaved and cooled to 50-60 °C,
then 1 ml 1 M CaCl2, 1 ml 1 M MgSO4 and 25 ml 1 M KH2PO4 are added). A bacterial lawn of
Escherichia coli OP50 strain grown in lysogeny broth (LB) medium (5 g yeast extract, 10 g
tripton, 10g NaCl and ddH2O to 1 l is autoclaved) is seeded on agar plates to serve as food
source for nematodes unless otherwise stated.
If the worms need to be stored for a longer period of time, they can be frozen. The
freezing process begins with the collection of L1 worms into 4 ml of S basal medium (50 ml 1
M pH6 KH2PO4, 5,8 g NaCl, 1 ml of 5 mg/ml cholesterol and H2O to 1 litre are autoclaved)
after which 4 ml of freezing solution (1.76 g NaCl, 2.04 g KH2PO4, 1.7 ml of 1 M NaOH, 71.6
ml glycerol and ddH2O to 300ml are mixed, autoclaved and 0.09 ml 1 M MgSO4 is added) is
added and mixed with the worms. Four aliquots of 1-1 ml are transferred into cryotubes,
which are thereafter kept at –70°C or in liquid nitrogen.
Keeping C. elegans strains clean is indispensable for the efficient genetic work. The
most common contaminations are different bacteria, mold and yeast. The easiest method of
cleaning a strain is to transfer the animals to a new, uncontaminated plate twice a day until
they lose the unwanted contaminations. For transferring the worms a chunk of agar can be
moved from one plate to another by a sterilized spatula. Another method is to pick single
animals with a worm picker (platinum wire in the tip of a Pasteur pipet).
The most effective way of purification is bleaching which means the treatment of
the washed animals with hypochlorite solution (2:1 mix of household bleach and 5 N NaOH),
which will kill the contaminant and all worms not protected by the egg shell. This method is
also used for egg preparation.
3.2 Genetic crossing, generating double mutants
Sexual dimorphism is characteristic for C. elegans, XX hermaphrodites and XO males
exist in the populations. Males arise by spontaneous X chromosome segregation failure, thus
their ratio is only about 0.05% in wild type populations. Hermaphrodites are able to fertilize
45
themself and lay approximately 300 eggs. Males can inseminate the hermaphrodites, which
will use male sperm preferentially (both types of sperm are stored in the spermatheca).
When a hermaphrodite is inseminated by a male, the number of the progeny can exceed
1,000. Propagation of self-fertilizating hermaphrodites allows the maintenance of invariant
genetic lines, while crossing hermaphrodites with males permits the establishment of
different genetic combinations.
The crossing in C. elegans is relatively simple: 10-15 males and about 10
hermaphrodites are put together on the same NGM plate to allow them to mate, and the
next day each hermaphrodite is transferred onto a new, separated plate. In order to cross
two lines, first N2 (wild type) males are generated for further crosses. Next, one of the two
strains (containing a mutation), which are planned to cross, is mated with N2 males and
finally the resulting heterozygous males (a/+; +/+) are crossed with the other mutants
(containing b mutation) to generate the a/+; b/+ trans-heterozygote. The wished double
mutants (a/a; b/b  1/16 ratio) can then easily be isolated in the F2 generation.
(http://wormbook.org/chapters/www_makingcompdmutants.2/makingcompdmutants.htm)
In the case when the mutation a lacks an obvious phenotype, it maybe
advantageous to find a genetic marker (e.g., lon – Long phenoype), which is located nearby
the mutation a. In this case, first lon/lon; b/b animals should be generated. Then a/a
hermaphrodites are mated with lon/+; b/+ males. non-lon; b/b descendants (a/a; b/b and
a/lon; b/b) of those F1 animals that also produce lon progeny (a/lon; b/+) are picked, and
those which do not produce lon F3 animals are the a/a; b/b.
lon-1(e185) mutation was used as marker for the following mutations during
crossings:
ced-4(n1162) distance: 0.8 cM
ced-4(n2273) distance: 0.8 cM
For genotyping deletional mutants simple technique exists called single worm
polimerase chain reaction (SWPCR). First, a primer pair should be designed flanking the
deletion. A single worm with unknown genotype should be lysed in single worm lysis buffer
(0.373 g KCl, 0.051 g MgCl2*6H2O, 0.121 g Tris, ddH2O to 99.1ml, 0.45 ml Tween 20 and 0.45
ml NP-40. pH is set to 8.3 and it is autoclaved) with proteinase K. Using the resulting lysate
as DNA template, the caracteristic sequence that contains or lacks the deletion is amplified.
46
Thus either wild-type or heterozygote or homozygous mutant can be identified by gel
electrophoresis.
SWPCR was used to identify the following mutations:
atg-18(gk378)
Left primer: 5’-TCCAGTGTCAGTGCTTCCTG-3’
Right primer: 5’-CTGTAAACGCTCGAGCAACA-3’
atg-7(tm2976)
SWPCR was used to identify this mutation after crossing.
Left primer: 5’-TCCTGCAGAATACGCAACAG-3’
Right primer: 5’-TGTCCGGTACCCTTCTTCTG-3’
From a lysate derived from a single worm it is also possible to detect a point
mutation by tetra primer ARMS-PCR (amplification refractory mutation system PCR). In this
method four primers are designed: two allele-specific primers called the inner primers and
the two outer primers. The 3’ ends of the inner primers are designed to the two allelic
variations from both sides. Thus, the two allele-specific amplicons are representing the two
alleles. By positioning the two outer primers at different distances from the polymorphic
nucleotide, the two allele-specific amplicons differ in length, allowing them to be separated
by gel electrophoresis.
Tetra primer ARMS-PCR was used to identify the following mutation:
ced-3(n717)
Inner left primer: 5’-TAAATGATAATTAATAAATTTTTGAAG-3’
Inner right primer: 5’-CTTCTTTCTCCACACTGGT-3’
Outer left primer: 5’-TTCCTAATGTAAACTGATATTTAATT-3’
Outer Right primer: 5’-TTAATTTTTTAAATGAGTTAACAATT-3’
3.3 RNAi treatment
In 1998, Fire and Mello discovered that injection of double stranded RNA (dsRNA)
into worms leads to specific degradation of the corresponding mRNA, a process termed RNA
interference (Fire et al., 1998). Soon afterwards, it was found that feeding worms on
bacteria engineered to produce dsRNA also induces a robust RNAi response (Timmons and
Fire, 1998). For this method, the HT115 E. coli strain is used carrying the feeding construct,
which contains a part of the gene of interest cloned into pPD129.36. In the vector pPD129.36
47
the desired sequence is flanked by two T7 RNA polymerase binding sites, which allow the
transcription of the insert in both sense and antisense directions. The expression of the T7
polymerase is induced by IPTG in HT115 bacteria. HT115 bacteria are used since they lack
RNAaseIII, which would be able to eliminate the produced dsRNA.
The engineered HT115 bacteria are grown in LB medium with antibiotics (HT115
strain is tetracyclin resistent, and pPD129.36 contains ampicillin resistence gene) and then
they are seeded onto RNAi plates (3 g NaCl, 2.5 g bacto-peptone, 17 g agar, 1 ml of 5 mg/ml
cholesterol and ddH2O to 1 l are mixed, autoclaved and cooled to 50-60 °C, then1 ml 1 M
IPTG, 0.5 ml 100 mg/ml ampicillin, 0.5 ml 50 mg/ml tetracyclin, 1 ml 1 M CaCl2, 1 ml 1 M
MgSO4 and 25 ml 1 M KH2PO4 are added). After animals are put onto the RNAi plates they
eat the bacteria and the gene of interest will be silenced.
The used construct that expresses bec-1 double-stranded RNA, contains 1.3 kb
fragment of bec-1 cDNA (yk391b8, Yuji Kohara clone) cloned into pPD129.36 vector.
3.4 Nematode strains and RNAi constructs
N2 wild type strain
MT2547 ced-4(n1162)III.
MT5816 ced-4(n2273)III.
MT1522 ced-3(n717)IV.
VC893 atg-18(gk378)V.
FX2976 atg-7(tm2976)IV.
CB369 unc-51(e369)V.
CB1189 unc-51(e1189)V.
EP.013 lgg-1(tm3489)II.
CB185 lon-1(e185)III.
BU43 unc-51(e1189)V.; lon-1(e185)III.
BU071 gfp::lgg-1 buIs1[GFP::plgg-1::LGG-1].
DP38 unc-119(ed3)III.
bec-1(RNAi)
48
3.5 Generated strains
EP.003 atg-18(gk378)V.; lon-1(e185)III.
EP.001 atg-18(gk378)/+V.; ced-4(n1162)III.
EP.005 atg-18(gk378)/+V.; ced-4(n2273)III.
EP.007 atg-18(gk378)V.; ced-3(n717)IV.
EP.004 unc-51(e369)V.; lon-1(e185)III.
EP.020 unc-51(e369)V.; ced-4(n1162)III.
EP.021 unc-51(e1189)V.; ced-4(n1162)III.
EP.022 atg-7(tm2976)/+IV.; lon-1(e185)III.
TIMI.005 atg-7(tm2976)/+IV.; ced-4(e1162)III.
EP.010 lgg-1(tm3489)/+II.; lon-1(e185)III.
EP.023 lgg-1(tm3489)/+II.; ced-4(n1162)III.
EP.002 gfp::lgg-1; ced-4(n1162)III.
BU311 unc-119(ed3)III.;pRH21[punc-119::unc-119]+bu81[pbec-1::GFP]
BU312 unc-119(ed3)III.;pRH21[punc-119::unc-119]+bu82[p(bZip∆)bec-1::GFP]
3.6 Determining lethality and brood size
For measuring embryonic and larval lethality about twenty adult hermaphrodites
were transferred to a clean NGM plate to lay eggs for two hours at room temperature
(synchronisation), then they were removed and the exact egg number was counted. After 24
and 48 hours both the number of dead eggs and larvae and also the amount of the survivals
were determined. The plates were kept at 20 °C during the experiment. Three parallel
experiments were carried out.
For determining the brood size, 10 adult worms were put on separated plates. All
the animals were transferred on a new plate every day, and the eggs layed from the previous
24 hours were counted on each plate. This was repeated until the animals were fertile.
Finally, the brood size of all hermaphrodites was summarized.
49
3.7 Bioinformatics and statistical analysis
We used ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and WormBlast
programs (wormbase.org) in order to find the appropriate transcription factor binding sites.
To determine lethality and brood size, three parallels of wild type, the appropriate
apoptotic and autophagic single mutants and the double mutant strains were analyzed. The
correspondence between replicates was tested by G probe.
We investigated whether the effect of different autophagic and apoptotic
mutations on the survival and brood size of the worm was independent. To address this
question, the three-way log linear modell was used.
3.8 Light and fluorescence microscopy
For taking high magnification images an Olympus BX51 light microscope was used.
Hot 4% agar droplet was put onto a glass slide and another one was placed onto it. Animals
were transferred onto the solidified agar in a drop of a solution containing 1:1 M9 medium
(3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl and ddH2O to 1 l are mixed, autoclaved and cooled to 5060 °C then 1 ml 1 M MgSO4 is added) and 1 M levamisole in order to paralyze the worms.
Finally, a glass coverslip was placed to the top of the agar.
Using Differential Interference Contrast (DIC) optics high quality Nomarsky images
could be obtained. DIC is an optical microscopy illumination technique used to enhance the
contrast in unstained, transparent samples and giving the appearance of a threedimensional physical relief corresponding to the variation of optical density of the sample,
emphasising lines and edges though not providing a topographically accurate image. Since
the density of the apoptotic corpses differ from the surrounding tissue, it is considerably
easy to detect them with this technique. They appear as a rounded object emerging from
the tissue.
For fluorescent images the light source was a mercury arc lamp and filters designed
for detecting the desired fluorescent marker were used (Table 2).
50
Table 2. Types and parameters of the used filters - For DAPI staining (blue), GFP labeling (green) and
TUNEL staining (red: TXRED) I chose the appropriate filter depending on its exciter and emission
wavelength.
filter name
U-MWU2
U-M41025
U-N41004
exciter filter
(nm)
BP 330-385
BP 450-490HQ
BP 532-592HQ
dichromatic
beamsplitter (nm)
DM 400
LP 495
LP 595
emission filter
(nm)
BA420
BP 500-530
BP 609-684
detected
staining
DAPI
GFP
TXRED
3.9 Measuring the level of apoptosis
Two different methods were used to determine the level of apoptosis in the
developing embryos: 1) counting the number of apoptotic corpses by DIC optics and 2) the
TUNEL assay. During the first technique images were taken from comma stage embryos at
different focal planes. Later the exact number of cell corpses was counted in each focal
plane (Fig. 2). In C.elegans, cells undergoing apoptosis display a refractile, raised button-like
appearance, which can be easily detected using Nomarsky optics.
TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling,
ROCHE) is a special method developed to detect and quantify apoptotic cell death at the
single-cell level in different tissues. During apoptosis, DNAse activity not only generates
double-stranded, low-molecular-weight DNA fragments (mono- and oligonucleosomes), but
also introduces strand breaks ("nicks") into the high-molecular-weight DNA. TUNEL reaction
identifies these events by labeling the free 3’-OH termini with terminal deoxynucleotidyl
transferase (TdT), which attaches fluorescently labeled nucleotides to all 3´OH-ends.
Combined with TUNEL, a DNA specific staining called DAPI (4',6-diamidino-2phenylindole) was also used. DAPI is a fluorescent dye that binds strongly to A-T rich regions
of the DNA.
First, eggs were prepared then embryos were placed in fixing solution (140 μl 2X
Witch’s Brew [400 μl NaCl, 8 ml KCl, 5 ml Na2EGTA, 5 ml Spermidin-HCl, 1.5 ml pH 7.4 NaPIPES and 25 ml methanol], 250 μl PFA (4%) and 250 μl 9:1 methanol-EDTA). After
incubation the sample was frozen in liquid nitrogen, melted and incubated again. Then it was
washed once with Tris-Triton buffer (5 ml pH 7.4 Tris-HCl , 500 μl Triton X-100 and 100 μl
EDTA) and twice with PBST-B buffer (1X PBS buffer [10X: 16 g NaCl, 0.4 g KCl, 0.4 g Na2HPO4,
0.4 g KH2PO4 and ddH2O to 200 ml], 0.1% BSA, 0.1 % Triton-X, 5 mM Sodium-azidand 1 mM
EDTA). At this step TUNEL reaction solution mixing with DAPI (100ng/ml) solution was added
51
to the sample and they were incubated together at 37 °C for an hour. Following this embryos
were washed twice with 1X PBS buffer and eggs were ready for analysis by fluorescent
microscopy.
3.10 EMSA (electrophoretic mobility shift assay)
Electrophoretic mobility shift assay (EMSA) is an important technique for
determining sequence-specific protein-DNA interactions. This assay is also referred to as gel
shift or gel retardation assay. During EMSA a mix of labeled oligonucleotides containing the
hypothetized binding sites and the potential DNA binding protein is loaded on nondenaturing polyacrylamide gel. The DNA-protein complex migrates more slowly than the
free DNA fragments during electrophoresis.
Full length recombinant ATF-2 and CES-2 proteins were purified with histamine
affinity chromatography. The interaction between these proteins and the newly identified
potential CES-2 regulatory region in the promoter of bec-1 was analysed on an 8% TBE-PAGE
native gel using an ethidium-bromide staining. In every reaction the CES-2 protein, ATF-2
protein and the oligonucleotides were used in a 5, 10 and 1 μM final concentration,
respectively. The protein-oligonucleotide mixtures were incubated at room temperature for
20 minutes before they were loaded on the gel.
3.11 Cloning of reporter constructs
A genomic fragment containing the 5’ regulatory region and the first two exons of
bec-1 was amplified from purified C. elegans genomic DNA using the following primers:
5’-CCCAAGCTTGGGAAAAATGCTCTTTATTGGAAAATTG-3’
5’-CGCGGATCCGCGGAGCATCCGAGATGAGCTTC-3’.
A bec-1::gfp construct was prepared by cloning the BamHI/HindIII digested PCR
product into pPD95.75 vector.
A QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used to generate the
mutated bZip_bec-1::gfp reporter construct. In the first step a PCR reaction was done using
the bec-1::gfp plasmid as DNA template and the following primers, which carry the
deletional mutation:
5’-CGAAGGCACTGAGCGACAGACGGATGGACG-3’
5’-CGTCCATCCGTCTGTCGCTCAGTGCCTTCG-3’.
52
In the next step the methylated, non-mutated parental DNA template was digested
with DpnI specific for methylated DNA. Thus, after digestion only the non-methylated PCR
product remains, which carries the desired mutation. This circular dsDNA was transformed
into XL10-GOLD supercompetent cells.
3.12 Generation of transgenic animals (microparticle
bombardment)
The unc-119(ed3) strain (paralyzed worms) was used to generate transgenic
animals. Young adult animals were grown on 5 large NGM plates (∅ 9 cm) seeded with the E.
coli strainNA22. Then, worms were washed with M9 solution, transferred to a new NGM
plate without bacteria and placed on ice.
The linearized DNA construct of interest, together with the linearized positive
selection marker containing a full length wild type unc-119 rescuing fragment, was bound to
gold micro particles (∅ 1 μm). Then the prepared animals were shot with the gold particles
covered with DNA by a BioRad PDS-1000/He high pressure vacuum system. After 4 days at
25 °C all moving, non-paralyzed animals should be selected as different transgenic lines.
Later, the F2 progeny of the moving F1 animals were screened for the presence of paralyzed
animals to determine whether the lines contain extra-chromosomal arrays or they are
integrated.
53
4 Results
4.1 Autophagy and apoptosis are redundantly required for C.
elegans development
4.1.1 Mutational inactivation of atg-18 in apoptosis deficient genetic
background causes full penetrant embryonic lethality
Although autophagy and apoptosis are essential mechanisms during the
development of several animal species, little is known about their interactions. To expand
our knowledge about this issue, I intended to perform a genetic analysis using C. elegans as a
modell system. To analyze the effect of autophagy and apoptosis on the viability of the
worms I created a series of double mutants defective in both autophagy and apoptosis. Then
I checked the rate of their lethality during embryogenesis and larval development compared
to that of wild type and the corresponding single mutants. First I crossed atg-18 mutants
with worms deficient in ced-3/ICE and ced-4/Apaf-1.
The ATG-18 protein is necessary for the recyclization of the autophagy proteins
from the membrane surface, thus it is crucial for the formation and growth of the new
autophagosomes (Nair et al., 2010). I used atg-18(gk378) which is a null allele carrying a 666
bp deletion that removes a portion of the 5’ regulatory region and the first 3 exons of atg18. The majority of the animals lacking atg-18 function are viable, fertile and their
morphology and behaviour is similar to that of wild type.
ced-4/Apaf-1 is one of the downstream genes of the canonical apoptosis pathway. It
participates in both caspase-dependent and –independent apoptotic cell death (Galluzzi et
al., 2008). In the experiments two different ced-4 alleles were used. The n1162 is a nonsense
point mutation containing a C/T substitution that results in an early ochre stop codon in the
first exon. ced-4(n1162) null mutants completely lack apoptosis (Ellis and Horvitz, 1986; Yuan
and Horvitz, 1992). n2273 is a hypomorphic allele carrying a G/A substitution, which leads to
altered splicing of the ced-4 mRNA. The ced-4(n2273) mutation causes a decreased apoptotic
activity compared to the wild type (Shaham and Horvitz, 1996). Most ced-4 deficient animals
are viable, fertile and they show wild-type morphology and behavior. The ced-3 gene, which
encodes a caspase, is the terminal effector of apoptosis in C. elegans. The hypomorphic n717
allele of ced-3 contains a G/A substitution in the 3’ splice site of the 7th intron (Shaham et
al., 1999). Most of these animals are also viable and fertile (Ellis and Horvitz, 1986).
54
I created and then analyzed atg-18(gk378); ced-4(-) and atg-18(gk378); ced-3(-)
double mutants, and found that after self-fertilisation atg-18(gk378)/+; ced-4(n1162)
heterozygous hermaphrodites produced viable homozygous atg-18(gk378); ced-4(n1162)
double mutant F1 progenies. However, their F2 descendants showed a full penetrant
embryonic lethality (Table 3, Fig. 15) atg-18(gk378); ced-4(n2273) and atg-18(gk378); ced3(n717) homozygous double mutants, F1 progeny of the corresponding heterozygous
hermaphrodites, were also viable, but their F2 descendants showed 36% and 47% embryonic
lethality, respectively. Most of the survivors died at the L1 larval stage and only 1-2% of the
animals reached the adulthood (Table 3, Fig. 15), which means a significant difference
compared to the phenotypes of the corresponding single mutants. The full penetrant
lethality of the atg-18(gk378); ced-4(n1162) double mutants, in comparison with the atg18(gk378); ced-4(n2273) worms, is caused by the used ced-4 null allele. The lower embryonic
lethality of atg-18(gk378); ced-3(n717) double mutants, compared to atg-18(gk378); ced4(n1162) animals, can be explained by the fact that ced-3(n717), in contrast to ced-4(n1162),
is a hypomorhic mutation.
Table 3. Mutational inactivation of atg-18 in apoptosis deficient background causes embryonic and
early larval lethality in the F2 generation.
genotype
number of number number
counted of dead of dead
embryos embryos L1 larvae
number of
animals
dead
dead L1
animals
reaching
embryos larvae
reaching
adulthood
(%)
(%)
adulthood
(%)
235
0.4
0.0
99.6
429
1.2
0.0
98.8
202
1.5
0.0
98.5
224
1.8
0.0
98.2
118
0.8
0.0
99.2
N2
atg-18(gk378)
ced-4(n1162)
ced-4(n2273)
ced-3(n717)
236
434
205
228
119
1
5
3
4
1
0
0
0
0
0
atg-18(gk378);
ced-4(n1162)
138
138
0
0
100.0
0.0
0.0
atg-18(gk378);
ced-4(n2273)
130
47
82
1
36.2
63.1
0.8
atg-18(gk378);
ced-3(n717)
224
106
106
12
47.3
47.3
5.4
55
Lethality (%)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
animals reaching
adulthood (%)
dead L1 larvae (%)
dead embryos (%)
Figure 15. Mutational inactivation of atg-18 in apoptosis deficient background causes embryonic
and early larval lethality - atg-18(gk378) mutants display a synthetic lehthal phenotype in ced4(n1162), ced-4(n2273) and ced-3(n717) mutant backgrounds. The embryonic lethality of atg-18
mutants in ced-4(-) null mutant background is fully penetrant. atg-18(gk378); ced-4(n2273) and atg18(gk378); ced-3(n717) double mutants produce a weaker phenotype; embryonic and larval lehality
also occur but with lower penetrance. N2 represents the wild-type strain.
atg-18(gk378), ced-4(n1162), ced-4(n2273) and ced-3(n717) single mutant embryos
displayed a nearly wild-type phenotype. In contrast, atg-18(gk378); ced-4(n1162) double
mutants underwent embryogenesis, but they exhibited severe morphological abnormalities
(Fig. 16). They displayed elongation defects, thus they could not lengthen and fold inside the
eggshell. In almost all cases the development of the pharynx and the gut was initiated, since
gut specific autofluorescent granules were present in the posterior part of the embryos (Fig.
16). These viable animals were able to move inside the egg shell, therefore functional
muscles should also have existed. Furthermore, in these embryos large necrotic-like vacuoles
appeared throughout the body (Fig. 16). A group of rounded cells, so called extruded cells,
were seen in several cases inside the eggshells, but clearly separated from the embryos.
56
Figure 16. Autophagy and apoptosis are redundantly required for the morphogenesis of C. elegans
– In comparison with the wild-type embryos (a) the atg-18(gk378); ced-4(n1162) double mutant
embryo (b) exhibits severe defects in elongation, body shape malformations and necrotic-like holes
(asterisk). The organs and the tissues start to develop, e.g., the pharynx (arrowhead) and the gut
precursor cells indicated by autofluorescent granules (arrows). A: anterior, P: posterior.
4.1.2 Inactivation of ced-4 enhances the embryonic and larval lethality of
unc-51 mutants
Next, different unc-51 mutant alleles and the ced-4 null allele were used for the
same type of experiment. Upstream autophagy modulator signals act via the unc-51/Atg1
gene product, as this protein is the key member of the autophagy initiator Atg1 complex. In
C. elegans, this gene, besides the regulation of autophagy, affects axonal vesicle transport,
which is essential for the normal development of the axons (Ogura et al., 1994; Toda et al.,
2008). I used two different mutations affecting unc-51. In the unc-51(e369) mutant, two
point mutations are localized in the unc-51 coding region: a silent mutation in the -1 opal
codon (TGA) to ochre (TAA), and an amber termination codon TAG in the CAG codon for
Q553. Thus the e369 allele might lead to production of a truncated protein, although this still
contains the kinase domain. In the coding region of the e1189 mutant allele, no mutation
was found. However, in worms homozygous for the e1189 allele, the unc-51 mRNA level is
decreased (32% compared to the wild type). mRNA level is also reduced in unc-51(e369)
57
worms (67% compared to wild type) (Ogura et al., 1994). Thus, the e1189 mutation could be
in the upstream regulatory region of unc-51, outside of the coding region, which is important
for transcription or mRNA stability. The presence of the mRNA which encodes the kinase
domain suggests that both unc-51 alleles used should contain hypomorphic mutations.
Although unc-51 reduction-of-function mutants are paralyzed due to the impaired axon
growth, most of these worms are viable and fertile. Their body length is smaller than that of
the wild type and this morphological deformity results from reduced cell size, which is a
consequence of decreased autophagic activity (Aladzsity et al., 2007). These animals can be
characterized by an egg-laying-defective phenotype as well.
According to my results, 14% and 4% of the unc-51(e369) single mutants died during
embryonic and larval development, respectively. Compared to these data, embryonic and
larval lethality of unc-51(e369); ced-4(e1162) double mutants was increased to 27% and
39%, respectively. Thus, 80% of the unc-51(e369) single mutants, and only 35% of the unc51(e369); ced-4(e1162) double mutants reached the adulthood (Table 4, Fig. 17).
Homozygous unc-51(e1189) single mutants displayed 15% embryonic and 15% larval
lethality. In turn, 39% of unc-51(e1189); ced-4(e1162) double mutants died as embryos and
45% of them showed larval lethality (Table 4, Fig. 17). The observed phenotype of animals
homozygous for the e1189 allele was more severe than that of the unc-51(e369) single
mutants, both in wild type and in ced-4 mutant background. This correlated with the
described mRNA levels in these mutants. Thus, according to my results the e1189 allele
seemed to be stronger than the e369 allele.
Table 4. Simultaneous inactivation of unc-51 and ced-4 resulted in embryonic and larval lethality
genotype
N2
ced-4(n1162)
unc-51(e369)
unc-51(e1189)
unc-51(e369);
ced-4(e1162)
unc-51(e1189);
ced-4(e1162)
number of number of number
counted
dead
of dead
embryos
embryos L1 larvae
number of
animals
reaching
adulthood
animals
dead
dead L1
reaching
embryos larvae
adulthood
(%)
(%)
(%)
236
205
70
107
1
3
10
16
0
0
3
16
235
202
57
75
0.4
1.5
14.3
15.0
0.0
0.0
4.3
15.0
99.6
98.5
81.4
70.1
140
38
54
48
27.1
38.6
34.3
62
24
28
10
38.7
45.2
16.1
58
100%
90%
Lethality (%)
80%
70%
60%
50%
40%
30%
20%
10%
0%
animals reaching
adulthood (%)
dead L1 larvae (%)
dead embryos (%)
Figure 17. Simultaneous inactivation of unc-51 and ced-4 resulted in embryonic and larval lethality
– Mutational inactivation of ced-4 in unc-51 reduction-of-function background significantly increases
the embryonic and larval lethality compared to the unc-51 single mutants.
unc-51(-); ced-4(-) double mutant worms, which died either during embryogenesis
or during postembryonic development, showed serious morphological defects (Fig. 18).
Although different tissues and organs started to develop, the correct, healthy shape of the
soma could not be formed. Invaginations were observed in both embryos and hatched
larvae. Positions of some organs in the double mutant larvae were also abnormal (Fig. 18).
59
Figure 18. unc-51(e369); ced-4(n1162) double mutant embryos (b) and L1 larvae (d) display severe
morphological defects, as compared tothe wild type (a, c) – Double mutant embryos can not
undergo body elongation. The presence of autofluorescence granules (indicated by white arrows)
suggests that gut cells are differentiated properly. The arrowhead in the mutant L1 larva shows the
abnormal position of the pharynx (d), as compared to the wild type (c). The asterisk indicates a
necrotic-like vacuole, theblack arrow points to an invagination in the posterior part of the animal. A:
anterior, P: posterior.
60
4.1.3 Inactivation of atg-7 and lgg-1 in ced-4 loss-of-function mutant
background causes embryonic lethality
I also generated atg-7(-); ced-4(-) and lgg-1(-); ced-4(-) double mutant animals to
confirm my previous data. ATG-7, which functions as an E1-like enzyme, is a member of both
ubiquitin-like conjugation systems, participating in the elongation and closure of the
autophagosome. It plays a role in the activation of LGG-1/Atg8, which is a microtubule
associated anchore protein and assists in the formation of autophagosomes and
autolysosomes (Ichimura et al., 2000).
The tm2976 allele of atg-7 was used during the analysis which contains an 11 bps
long insertion and a 262 bps deletion in the 3rd and 4th exon. Animals homozygous for this
allele displayed full penetrant embryonic and early larval lethality in the F2 generation.
Although this allele has not been characterized yet, based on the severe phenotype it
causes, tm2976 might be a genetic null or a strong hypomorphic mutation.
In the case of lgg-1, the tm3489 deletional allele was used, which removes 175 bps
from the first exon. According to the sequence analysis of the mutant allele, the deletion
localizes in the first part of the ORF, removing a major part of the gene, and causes
frameshift resulting in a premature stop codon in the residual part of the sequence.
Homozygous lgg-1(tm3489) mutant worms also produced 100% embryonic or larval lethality
in the F2 generation. Thus, tm3489 may represent a genetic null allele of lgg-1. Both atg7(tm2976) and lgg-1(tm3489) single mutants showed a more severe phenotype than that of
atg-18(gk378), unc-51(e369) and unc-51(e1189) autophagic mutant animals.
After generation of atg-7(tm2976); ced-4(n1162) and lgg-1(tm3489); ced-4(n1162)
double mutants, I measured their viability compared to the corresponding single mutants.
Homozygous mutants in the F1 generation, progeny of both the atg-7(tm2976)/+ and lgg1(tm3489)/+ heterozygous were viable and fertile. On the contrary, 12% and 4% of the F2
homozygous atg-7(tm2976) and lgg-1(tm3489) single mutants died during embryonic
development, respectively, and the hatched larvae showed a full penetrant lethality (Table 5,
Fig. 19). Accordingly, the F1 generation of atg-7(tm2976); ced-4(n1162) and lgg-1(tm3489);
ced-4(n1162) double mutants were also viable, but all of their offsprings died already during
embryogenesis (Table 5, Fig. 19).
61
Table 5. Inactivation of either atg-7 or lgg-1 genes in ced-4 null mutant background causes full
penetrant embryonic lethality in the F2 generation
number of number of number
counted
dead
of dead
embryos
embryos L1 larvae
genotype
N2
ced-4(n1162)
atg-7(tm2976)
lgg-1(tm3489)
atg-7(tm2976);
ced-4(n1162)
lgg-1(tm3489);
ced-4(n1162)
number of
dead
dead L1
animals
embryos larvae
reaching
(%)
(%)
adulthood
animals
reaching
adulthood
(%)
236
205
335
79
1
3
41
3
0
0
294
76
235
202
0
0
0.4
1.5
12.2
3.8
0.0
0.0
87.8
96.2
99.6
98.5
0.0
0.0
112
112
0
0
100.0
0.0
0.0
65
65
0
0
100.0
0.0
0.0
100%
90%
Lethality (%)
80%
70%
60%
50%
40%
30%
20%
10%
0%
animals reaching
adulthood (%)
dead L1 larvae (%)
dead embryos (%)
Figure 19. Inactivation of either atg-7 or lgg-1 genes in ced-4 null mutant background causes full
penetrant embryonic lethality - Although most of the atg-7(tm2976) and lgg-1(tm3489) single
mutant worms show L1 arrest, 100% of the atg-7(tm2976);ced-4(n1162) and lgg-1(tm 3489);ced4(n1162) double mutant animals die during embryonic development.
The phenotype of atg-7(tm2976); ced-4(n1162) and lgg-1(tm3489); ced-4(n1162)
double mutant embryos (Fig. 20) were similar to that of atg-18(gk378); ced-4(n1162) double
mutants (Fig. 16). They were also unable to elongate and showed severe body
malformations. Abnormal changes occured during tissue and organ development, and huge
necrotic-like vacuoles appeared inside the animals (Fig. 20). The formation of the pharynx
and the gut was also initialized, indicating that the specification of several cell types occured
62
normally. Cell aggregates detached from the embryo that die in the extraembryonic space of
the egg were also present (indicated by the red dotted line in Fig. 20b).
Figure 20. Autophagy and apoptosis are redundantly required for normal embryogenesis – While a
wild-type embryo (a) elongates and folds properly inside the eggshell, these morphogenetic events
can not take place in an atg-7(tm2976); ced-4(n1162) (b) mutant embryo. Organ and tissue
development are iniciated in the double mutants as well. Arrowheads show the pharynx and arrows
show autofluorescent granules of the gut. The asterisk indicates a necrotic-like vacuole in the mutant
embryo. Extraembryonic cells appear inside the eggshell of the double mutant embryo (enclosed by
the dashed line). A: anterior, P: posterior.
4.1.4 Silencing of bec-1 decreases viability and causes sterility in apoptosis
deficient mutants
Finally, I examined the effect of bec-1 RNAi treatment on the viability of ced-4(-) and
ced-3(-) mutant worms. The BEC-1/Atg6 protein has a crucial role in the vesicle nucleation
step of autophagy. bec-1 influences the viability, normal growth rate, movement and body
size of C. elegans, and it also participates in the morphogenesis of the vulva. Mutational
inactivation of bec-1 leads to embryonic lethality (Takacs-Vellai et al., 2005).
Silencing of bec-1 by RNAi resulted in a fertile F1 generation, with only a 10%
embryonic lethality (Table 6, Fig. 21). Thus, bec-1(RNAi) animals slightly phenocopy the null
mutant phenotype of bec-1(-) worms. When ced-4(n2273) and ced-3(n717) mutants were
treated with bec-1 dsRNA, half of the F1 progeny died as embryos, the other half reached
63
the adulthood, but became sterile (Table 6, Fig. 21). Thus, inactivation of apoptosis in bec-1
silenced background affects both embryonic development and fertility of adult
hermaphrodites.
Table 6. Silencing of bec-1 by RNAi in ced-4(-) and ced-3(-) mutant background causes increased
embryonic lethality and sterility
number of number number number
counted
of dead of fertile of sterile
embryos embryos adults
adults
genotype
N2
ced-4(n2273)
ced-3(n717)
bec-1(RNAi)
ced-4(n2273);
bec-1(RNAi)
ced-3(n717);
bec-1(RNAi)
dead
embryos
(%)
fertile
adults (%)
sterile
adults (%)
236
228
119
683
1
4
1
77
235
224
118
606
0
0
0
0
0.4
1.8
0.8
11.3
99.6
98.2
99.2
88.7
0.0
0.0
0.0
0.0
412
240
0
172
58.3
0.0
41.7
390
189
0
201
48.5
0.0
51.5
100%
90%
Lethality (%)
80%
70%
60%
50%
40%
30%
20%
10%
0%
sterile adults (%)
fertile adults (%)
dead embryos (%)
Figure 21. Silencing of bec-1 by RNAi in ced-4 and ced-3(-) mutant background causes embryonic
lethality and sterility – bec-1 RNAi treatment significantly increases the lethality of ced-4(-) and ced3(-) mutants, compared to the wild type, and all of the animals reaching adulthood become sterile.
64
4.1.5 Simultaneous inactivation of autophagy and apoptosis causes a
synthetic brood size-reduction phenotype in C. elegans
I also analyzed whether mutations in autophagy and apoptosis genes separately or
together had any effect on the brood size of the nematode. A wild-type C. elegans individual
produces approximately 300 progeny at 20 °C. Basically, the brood size of the self-mating
hermaphrodite corresponds to the number of sperms produced prior to the rate of the
oogenesis. However, mutations affecting the quality or quantity of the oocytes also reduce
the number of progeny.
I found that the average brood size of atg-18 deficient animals was reduced by 15%,
compared to the wild type. ced-4(n2273) hypomorphic mutant worms, which are partially
deficient for apoptosis, and ced-4(n1162) null mutants completely lacking apoptosis
displayed a similar effect on the number of eggs layed. The ced-3(n717) mutation seemed
not to influence the brood size of theworms (Table 7, Fig. 22). atg-18(gk378); ced-4(n1162),
atg-18(gk378); ced-4(n2273) and atg-18(gk378); ced-3(n717) double mutants produced
approximately 53, 35 and 27% lessembryos, respectively, compared to the wild type (Table
7, Fig. 22). Thus, parallel inactivation of autophagy and apoptosis causes a significantly
higher reduction in brood size in comparison with that of the corresponding single mutants.
Table 7. Simultaneous inactivation of atg-18 and apoptotic genes results in a more severe
reduction of the brood size than inactivation of either single mutants.
genotype
289.3
245.4
237.2
243.8
284.1
average brood size
compared to the wild
type (%)
100.0
84.8
82.0
84.3
98.2
135.5
46.8
188.5
65.2
212.0
73.3
average brood size
N2
atg-18(gk378)
ced-4(n1162)
ced-4(n2273)
ced-3(n717)
atg-18(gk378);
ced-4(n1162)
atg-18(gk378);
ced-4(n2273)
atg-18(gk378);
ced-3(n717)
65
350
Average brod size (pcs)
300
250
200
150
100
50
0
N2
ced-4(n2273)
atg-18(gk378);ced-4(n2273)
atg-18(gk378)
ced-3(n717)
atg-18(gk378);ced-3(n717)
ced-4(n1162)
atg-18(gk378);ced-4(n1162)
Figure 22. Simultaneous inactivation of atg-18 and apoptotic genes results in a synthetic reduction
of the brood size – The brood size of atg-18, ced-4 and ced-3 single mutant animals is smaller than
that of the wild type. Furthermore, the progeny number of atg-18(gk378); ced-4(n1162), atg18(gk378); ced-4(n2273) and atg-18(gk378); ced-3(n717) double mutants are significantly reduced in
comparison with those of either single mutants. Bars indicate S.E.M.
4.1.6 The level of apoptosis increases in autophagic mutant background
Synthetic lethality in double mutants defective for both apoptosis and autophagy
raised the intriguing possibility that the two cell killing mechanisms can replace each other
when one of them is missing. Accordingly, the level of apoptosis should be increased in
autophagic defective mutants and vica versa, the rate of autophagy should be elevated in
apoptosis deficient mutant background.
We used two different techniques to investigate apoptotic activity in embryos
lacking the activity of an autophagy gene. First, I counted the number of apoptotic cell
corpses using Nomarsky optics in autophagic mutant background.
Although 131 cells die by apoptosis during normal embryonic and larval
development of a wild-type hermaphrodite, because of the relatively fast removal of dead
cells only a few cell corpses can be seen at a certain time point. According to this, we saw
only one or two corpses in a wild-type comma stage embryo. At the same time, the average
number of the observed apoptotic events in embryos lacking lgg-1 activity is 4 (Table 8.).
66
Table 8. The number of apoptotic cell corpses elevates in lgg-1 mutants.
genotype
number of
apoptotic
corpses
number of corpses
relative to wild type
(%)
N2
1.5
100
lgg-1(tm3489)
3.9
260
Second, we determined the number of corpses using the apoptosis-specific TUNEL
assay. The TUNEL reaction identifies and labels DNA nicks generated in the nuclei of
apoptotic cells, by attaching fluorescently labeled nucleotides to the free 3´OH-ends. Thus,
cells undergoing apoptosis can be identified by fluorescent microscopy. We also used DNAspecific DAPI staining combined with TUNEL in order to see the individual nuclei and to
assess the stage of embryos.
Using this method, we counted an average 1.3 fluorescent signs indicating apoptotic
events in wild-type embryos (Table 8). The average number of TUNEL positive cells was more
than two times higher in atg-18(gk378) and unc-51(e369) mutant embryos than in wild-type
embryos (Table 9, Fig. 23). Although the standard deviation of data measured by TUNEL was
high in all cases, the differences were still significant. We conclude that both methods
showed that autophagic mutants display increased apoptotic activity.
Table 9. The number of the TUNEL positive cells increases in autophagy mutant background.
genotype
N2
atg-18(gk378)
unc-51(e369)
number of
TUNEL positive
cells
1.3
2.7
2.8
67
TUNEL positive
cells (%)
100
211
219
e
4,5
Number of TUNEL positive cells
4
3,5
3
N2
2,5
atg-18(gk378)
2
unc-51(e369)
1,5
1
0,5
0
Figure 23. The number of TUNEL positive cells increases in autophagy mutant background – A wildtype embryo stained with TUNEL (b) shows a single apoptotic cell (the white arrow), while an atg-18
mutant displays three TUNEL positive cells (d; white arrows). DAPI staining of the wild-type (a) and
the atg-18(-) mutant embryo (c) shows that they are in a similar stage of embryogenesis. (e) atg-18
null mutants and unc-51 hipomorphic mutants display elevated levels of TUNEL positive cells. Bars
indicate S.E.M.
4.1.7 Autophagic activity does not change in apoptotic mutant background
Next, I monitored autophagic activity in apoptosis deficient mutant background. I
used LGG-1/Atg8 as a marker specific for autophagy, since LGG-1 is present on the surface of
both autophagosomes and autolysosomes for a long period of time. A transgenic line
carrying an integrated plgg-1::gfp::lgg-1 construct was applied for this analysis (Fig. 24).
I crossed the gfp::lgg-1 strain with the ced-4(n1162) apoptotic null mutant one. This
construct was expressed at a basal level in every cell during development, and showed a
dotted pattern (i.e., foci) in the cytoplasm. I checked lgg-1 expression in ced-4(n1162)
mutant background at different developmental stages, but I could not detect any difference
in the pattern or expression level of the transgene compared to wild type (Fig. 25).
68
Figure 24. The structure of the gfp::lgg-1 construct – The GFP marker is fused to the N terminus of
the LGG-1 protein. Approximately, 1.7 kb of DNA sequence is used from the 5’ upstream regulatory
region of lgg-1.
a
P
A
d
A
P
b
A
P
c
P
A
e
P
f
P
A
A
Figure 25. The expression level of gfp::lgg-1 does not change in apoptosis deficient background – In
a wild-type embryo, gfp::lgg-1 shows a punctated expression pattern in all cells (a-DIC-, bfluorescent-, c-merged image). In a ced-4 mutant embryo, this construct displays the same
expression pattern (d-DIC-, e-fluorescent-, f-merged image). A: anterior, P: posterior.
69
4.2 Autophagy and apoptosis are transcriptionally co-regulated in
C. elegans
4.2.1 The promoter region of bec-1 contains a CES-2/ATF-2 binding site
As we saw that autophagy and apoptosis are redundant processes during C. elegans
embryogenesis, we assumed that common regulatory mechanisms governing both systems
should exist. Therefore, we searched for apoptosis-related transcription factors which could
regulate autophagy genes as well. Using bioinformatics, we analyzed the 5’ regulatory
regions of autophagy genes, and found a conserved binding site for the CES-2 (cell death
specification-2) transcription factor approximately 400 base pairs upstream of the
translational initiation site of the bec-1 gene. The conserved binding site of CES-2 is defined
A
C
by the /G T T A C G T A A /T sequence logo (Wang et al., 2006). Since bec-1 orthologs exist
in closely related Caenorhabditis species, we examined by a ClustalW-based sequence
comparison whether they also contain the potential binding site. After analysing the
corresponding C. remanei and C. briggsae genomic regions, we found that they display only
minor changes in these sites, compared to that of C. elegans (Fig. 26). Thus, the potential
CES-2 binding site in the upstream regulatory region of bec-1 is conserved.
ces-2 encodes a basic leucin zipper like (bZip-like) protein (Wang et al., 2006). The
bZip transcription factors contain a conserved leucin zipper dimerisation domain and a basic
DNA-binding domain. Homodimers and heterodimers with other bZip factors also exist
(Metzstein et al., 1996). They play a major role in development, oncogenesis and regulate
the circadian rhythm.
In C. elegans, CES-2 plays a major role in the elimination of NSM sister cells. The
NSM precursors differentiate into serotonergic neurons and the NSM sisters. In the
serotonergic neurons, CES-1 promotes survival by downregulating egl-1. On the contrary, in
NSM sister cells CES-2 induces apoptotic cell death by activating egl-1 via the inhibition of
ces-1 (Metzstein and Horvitz, 1999; Thellmann et al., 2003) (see chapter 1.2.4 Regulation of
apoptosis).
CES-2 also regulates the morphological and physiological features of the excretory
duct cell, together with its paralog cAMP-dependent transcription factor-2 (atf-2), another
bZip-like transcription factor (Wang et. el., 2006). They share a common binding site and
transcriptionally activate the gene lin-48 (abnormal cell lineage-48), which is required for the
70
development of the excretory duct cell. While ces-2 is expressed only during embryogenesis,
atf-2 is also active during larval development. The function of ATF-2 looks more essential
than that of CES-2, since 80% of the atf-2(-) mutants die as embryos in contrast to ces-2(-)
mutants which do not display embryonic lethality (Wang et al., 2006).
Figure 26. The bZip-like binding site in the promoter of bec-1 is conserved among different
Caenorhabditis species – The potencial target sequence of CES-2/ATF-2 is located about 400 bp
upstream from the ATG start codon of bec-1 in C. elegans, C. remanei and C. briggsae. There are only
minor differences between the three binding sites. Red color indicates nucleotides that are
conserved in the consensus site, black nucleotides show differences from the consensus sequence,
non-conserved nucleotides are blue, conserved flanking nucleotides are indicated in green.
4.2.2 CES-2 and ATF-2 can bind to the promoter region of bec-1 in vitro
According to my previous results and the data above, we hypothetized that ATF-2
and CES-2 transcriptionally modulate the activity of the bec-1 gene. To investigate whether
the potential binding site resulted from the bioinformatic analysis is functional, we
performed a gel retardation assay (EMSA or Electrophoretic Mobility Shift Assay).
71
Table 10. Binding sites of the ATF-2 and CES-2 transcription factors - The table shows the consensus
sequence (1st row) and the bZip-like binding site located in the regulatory region of lin-48 (2nd row)
and bec-1 (3rd row), as well as the sequence of the mutated binding site of the bec-1 promoter (4th
row). Green indicates the consensus bases, yellow marks the differences from it and red shows the
substitutions introduced into the bec-1 regulatory region to destroy the binding site.
consensus
A/G
T
T
A
C
G
T
A
A
C/T
lin-48
A
T
T
A
C
C
C
A
A
C
bec-1
A
C
T
A
C
G
T
A
A
C
bec-1mut
A
C
A
A
T
G
T
T
G
T
Constructs for CES-2 and ATF-2 were kindly provided by Helene Chamberlain (Wang
et al., 2006). The 11.2 kDa CES-2 and the 8.8 kDa ATF-2 bZip domains were expressed in E.
coli strains BL21 and BL21 Codonplus (Stratagene) cells. The oligonucleotides, which were
synthetized for the experiment, contained the potential bZip-like transcription factor binding
site. A second type of oligonucleotide was also produced, in which crucial positions of the
binding site were mutated in order to confirm specificity (Table 10).
Figure 27. ATF-2 and CES-2 bind to a conserved binding site in the regulatory region of bec-1 in
vitro - ATF-2 is able to bind to a putative binding site of bec-1 in vitro (d), while it cannot bind to it
when the target sequence was previously mutated (f). CES-2 can also bind to the oligonucleotides if
they contain the candidate site (j), and there was no interaction in its absence (l). lin-48
oligonucleotides are used as a positive control (b and h). The upper bands from the gels are missing
in all cases when oligos are loaded without proteins (a, c, e, g, i and k).
In the gel shift assay, oligonucleotides containing the bZip-like binding site of lin-48
were used as positive control, based on the study of Wang and coworkers (2006). Both the
72
expressed ATF-2 and CES-2 bZip domains were able to bind this control (Fig. 27b, h). These
bindings were indicated by the upper bands on the gel consisting of protein-DNA complex,
which migrates slower than the free oligos. On the gels, the lower bands show the free
oligonucteotides. As a negative control of the system, only the oligos were loaded on the gel
without proteins. In these cases the corresponding upper bands were missing (Fig. 27a, g).
Next, oligos containing the potential bec-1 bZip-like binding site were mixed with either
ATF-2 or CES-2, and loaded on the gel. On lanes d and j, the upper bands indicate bindings
with both bZip domains (Fig. 27d, j), and the corresponding upper bands were missing in the
absence of loaded proteins (Fig. 27c, i). Interestingly, the binding of ATF-2 to the bec-1
promoter element was stronger than its interaction with the positive control lin-48 oligo.
Introducing mutations into key positions of the bZip-like binding site in the bec-1 promoter
fragment led to the lack of DNA-protein interaction with both bZip-like domains (Fig. 27f, l).
The corresponding bands were missing when only these mutated oligos were loaded on the
gel without the expressed transcription factor domains (Fig. 27e, k). This indicates that both
ATF-2 and CES-2 bZip-like domains are able to bind the candidate binding site located in the
promoter of bec-1 in vitro, and these interactions are sequence specific.
4.2.3 The bZip-like binding site is crucial in the regulation of bec-1
As both ATF-2 and CES-2 were able to bind specifically to the potential binding site
in the regulatory region of bec-1 in vitro, we intended to examine whether this sequence is
functional in vivo. To this end we generated a construct containing a 2.5 kbp promoter
region of bec-1, carrying the potential CES-2/ATF-2 binding site, and the first two exons
fused to gfp. Next we mutated this construct by deleting the entire binding site in order to
investigate its specificity (Fig. 28). Finally, we generated stable integrated transgenic
nematode strains carrying either the wild-type or the mutant construct.
73
Figure 28. Scheme for two bec-1transcriptional reporters - In the wild-type sequence (bec-1::gfp),
red color indicates the nucleotides of the potential ATF-2/CES-2 binding site. In the mutated
construct (bZip∆_bec-1::gfp), blurred box indicates the nucleotides that were deleted from the wildtype reporter.
We analyzed and compared bec-1::gfp expression of the created lines in three
developmental stages: at the embryonic comma stage, and at the L2 and L4 larval stages. In
all stages examined, we could observe a significant increase in the expression of the mutated
bec-1::gfp construct, as compared to the wild type. Both anterior and posterior parts of
comma stage embryos showed elevated expression of the mutant construct (Fig. 29). In L2
larvae, hypodermal seam cells and some head neurons showed expression of the wild-type
construct. Deletion of the CES-2/ATF-2 binding site resulted in increased expression of bec-1
in all tissues examined in L2 larvae (Fig. 30). At the L4 larval stage, bec-1 was expressed in
the developing vulva, and we could detect a significant change in the expression of the
mutated construct, in comparison with the wild type in this organ (Fig. 31). Thus, it seems
that ATF-2 and/or CES-2 can specifically repress bec-1 through this binding site in several
developing tissues.
74
a
A
d
g
100%
A
90%
P
b
P
A
e
P
c
A
P
A
f
Ratio of the embryos (%)
80%
70%
high expression
60%
intermediate
expression
50%
40%
low expression
30%
A
20%
10%
0%
P
bec-1::gfp
P
bZip∆_bec-1::gfp
Figure 29. The bZip-like binding site influences the expression level of bec-1::gfp in embryos - As a
result of the absence of ATF-2/CES-2 binding site, bZip∆_bec-1::gfp is upregulated in several cells of a
comma stage embryo (d-DIC-, e-fluorescent-, f-merged image), compared to the bec-1::gfp wild-type
reporter (a-DIC-, b-fluorescent-, c-merged image) at the same developmental stage. All of the
fluorescence images were taken with the same exposition time. The diagram (g) shows the
percentage of embryos with different bec-1 expression. A: anterior, P: posterior.
a A
100%
d A
90%
P
80%
e
A
P
P
c A
f A
Ratio of the larvae (%)
P
b A
g
high
expression
70%
60%
intermediate
expression
50%
40%
low
expression
30%
20%
10%
P
P
0%
bec-1::gfp
bZip∆_bec-1::gfp
Figure 30. The bZip-like binding site influences the expression level of bec-1::gfp in early-stage
larvae – The expression level of the bec-1::gfp lacking the ATF-2/CES-2 binding site is elevated in
some head neurons and in the hypodermal seam cells (d-DIC-, e-fluorescent-, f-merged image), as
compared to the wild type (a-DIC-, b-fluorescent-, c-merged image). All of the fluorescence images
75
were taken with the same exposition time. The diagram (g) shows the percentage of larvae with
different bec-1 expression. Arrows show hypodermal seam cells. A: anterior, P: posterior.
a
d
g
100%
90%
80%
high
expression
b
e
Ratio of the vilvae (%)
70%
60%
intermediate
expression
50%
40%
low
expression
30%
c
f
20%
10%
0%
bec-1::gfp
bZip∆_bec-1::gfp
Figure 31. The bZip binding site influences the expression level of bec-1::gfp in the L4-stage vulva
cells – bZip∆_bec-1::gfp, lacking the ATF-2/CES-2 binding site (d-DIC-, e-fluorescent-, f-merged image)
is excessively expressed in the vulval tissue at the L4 larval stage, in comparison with the wild-type
bec-1::gfp construct (a-DIC-, b-fluorescent-, c-merged image). All of the fluorescence images were
taken with the same exposition time. The diagram (g) shows the percentage of larvae with different
bec-1 expression levels in the vulva tissue. Arrowheads indicate the developing vulvae.
76
5 Discussion
5.1 Interplay between apoptosis and autophagy
Because of the importance of each process alone, the interplay between apoptosis
and autophagy is intensively studied, but our knowledge is still highly limited about their
regulatory network. There are three different types of link between the two processes,
although each of them applies to a certain cell type, stimulus, and environment: i) first, both
apoptosis and autophagy can act as partners to induce cell death in a coordinated or
cooperative manner, ii) second, autophagy supports apoptosis, participating in certain
morphological and cellular events that occur during apoptotic cell death, but does not lead
to death by itself, iii) third, autophagy can act as an antagonist to block apoptotic cell death
by promoting cell survival. The regulatory network of apoptosis and autophagy seems to be
complex since they interact at different levels (Eisenberg-Lerner et al., 2009).
i) An example for the cooperative relationship of the two processes was observed in
light-damaged retinas and in oxidative stress induced photoreceptor cells in mice
(Kunchithapautham et al., 2007). Inhibition of either autophagy or apoptosis partially
rescued cell survival. Furthermore, autophagy was shown to be necessary for the induction
of apoptosis. Interestingly, simultaneous inhibition of both pathways resulted in increased
cell death by necrosis.
ii) An example for the apoptosis supporting role of autophagy is the
phosphatidylserine exposure, which provides the ‘eat me’ signal to neighboring phagocytes
and hence mediates cell engulfment and clearance of apoptotic bodies. During nutrient
deprivation, autophagy is required for the maintenance of cellular ATP levels which enables
the exposure of these signal molecules. Absence of autophagy leads to impaired clearance of
apoptotic cell corpses during embryonic development (Qu et al., 2007).
iii) To antagonize apoptosis, autophagy protects non-transformed epithelial cells
from apoptotic cell death induced by the loss of matrix attachment (Fung et al., 2008).
Additionally, autophagy maintains genomic integrity of the cell under metabolic stress, or
radiation damage (Mathew et al., 2007; Paglin et al., 2001). In the absence of autophagic
activity, metabolic stress leads to increased DNA damage, gene amplification and
chromosomal abnormalities, which activate the apoptotic pathway. These survival
mechanisms are caused by the elimination of depolarized mitochondria that are a source for
77
genotoxic ROS (Kim et al., 2007). Furthermore, clearance of damaged mitochondria may
directly restrain apoptosis by preventing MOMP and the release of pro-apoptotic molecules
such as cytochrome C and Smac/Diablo (Kim et al., 2007).
At the molecular level, the Bcl-2 family members are probably the best-known
regulators of both autophagy and apoptosis. Bcl-2 is an inhibitor of apoptotic cell death
(Vaux et al., 1988). It antagonizes the pro-apoptotic activity of Bax/Bak and blocks MOMP,
thereby preventing apoptosis (see chapter 1.2.3. The mechanism of apoptosis in mammals
and Fig. 5). Several Bcl-2 family members, like Beclin 1, activate autophagy (Liang et al.,
1999). Beclin 1 binds to Bcl-2/Bcl-XL through a BH3 domain that mediates docking to the
BH3-binding groove. The binding of Bcl-XL to Beclin 1 interferes with the Class III
phosphatidylinositol 3-kinase complex formation (Noble et al., 2008). Signals that disrupt the
constitutive Bcl-2/Bcl-XL–Beclin 1 association promote autophagy. For example, Beclin 1 is
phosphorylated by DAPk on the BH3 domain, which reduces its affinity to Bcl-XL (Zalckvar et
al., 2009). In the reciprocal manner, JNK phosphorylates Bcl-2, triggering its release from
Beclin 1 following starvation-induced autophagy (Wei et al., 2008). A second mechanism
leading to the dissociation of the complex involves the competitive replacement of Beclin 1
by other BH3-containing proteins, such as BNIP3 (Zhang et al., 2008). Bif-1, a Beclin 1
interacting protein that is a member of the endophilin B family, also regulates apoptosis by
binding and/or activating Bax/Bak (Takahashi et al., 2005).
5.2 Autophagy and apoptosis are necessary together for the
viability of C. elegans
Although preliminary results indicate that autophagy and apoptosis are essential
cellular processes during the development of several animal species, the nematode C.
elegans seems to develop normally in the absence of either of the processes. I hypothesized
that both mechanisms are important during development in spite of the superficially normal
morphology of the single mutants. Furthermore, this model provides an opportunity to
create autophagy-apoptosis double mutants in order to investigate the interplay between
them. According to our present knowledge, atg-18 is the only autophagy-specific gene in C.
elegans, thus the developmental functions of autophagy can be investigated by the
inactivation of this gene. Moreover, I also worked on unc-51/Atg1, atg-7, lgg-1/Atg8
autophagy mutants and bec-1 RNAi treated animals. These genes are multifunctional, since
78
they play roles in diverse cellular mechanisms (e.g. endocytosis, membrane transport, etc.;
Okazaki et al., 2000; Kim et al., 1999), although all of them participate in autophagy as well
(Aladzsity et al., 2007; Toth et al., 2008).To analyze apoptotic cell death , I used hypomorphic
ced-4, and ced-4 null mutant animals. Additionally, ced-3 mutants were applied.
I analyzed the viability of atg-18(gk378); ced-4(n1162), atg-18(gk378); ced4(n2273), atg-18(gk378); ced-3(n717), unc-51(e369); ced-4(n1162), unc-51(e1189); ced4(n1162), atg-7(tm2976); ced-4(n1162) and lgg-1(tm3489); ced-4(n1162) homozygous
double mutant animals. First, I generated strains which were homozygous for the apoptotic
mutant gene and heterozygous for the corresponding autophagy gene. They all produced
viable and fertile homozygous double mutants in the F1 progeny. In contrast, most of the F2
descendants of these F1 homozygous double mutants died during embryogenesis, or
reached but could not pass the L1 larval stage. The survival of the F1 generation, in contrast
to the lethality of the F2 progeny, can be explained by maternal contribution of the
examined autophagy genes.
Indeed, it is known that autophagy functions during early embryogenesis, and
mRNA products of autophagy genes are maternally loaded into mammalian oocytes
(Tsukamoto et al., 2008). Also, in our experiments, atg-7(tm2976) and lgg-1(tm3489) alleles
seem to be maternal-effect mutations, since the F1 progeny of the corresponding
heterozygotes are superficially wild-type, but their F2 progeny arrest development at the L1
stage (Fig. 19). Thus, autophagy genes are maternally affected in C. elegans, too. There is no
published data proposing that ced-4/Apaf-1 and ced-3/ICE caspase would be maternal-effect
genes. To investigate whether apoptosis also contributes to maternal effect lethality of the
autophagy-apoptosis double mutants, the following experiment should be carried out:
counting embryonic lethality of homozygous double mutant F1 and F2 progeny of worms
which are homozygous for an autophagic and heterozygous for an apoptotic mutation.
atg-18(gk378); ced-4(n1162), atg-7(tm2976); ced-4(n1162) and lgg-1(tm3489); ced4(n1162) double mutant strains displayed a full penetrant embryonic lethal phenotype (Fig.
15, 19). On the contrary, in unc-51(e369); ced-4(n1162), unc-51(e1189); ced-4(n1162) double
mutants embryonic lethality was only 27% and 39%, respectively, and 39% and 45% of the
animals could reach the early larval stage but could not pass it (Fig. 17). mRNA is transcribed
from both the e369 and the e1189 allele of unc-51, and these mutations do not affect the
kinase domain of UNC-51. Furthermore, it has been shown that hypodermal seam cells of
79
both unc-51(e369) and unc-51(e1189) mutants still contain autophagic vesicles even if they
are morphologically deformed (Aladzsity et. al., 2007). Thus, e369 and e1189 shoud be
hypomorphic mutations. This can explain the observed weaker phenotype of unc-51(-); ced4(e1162) double mutants, in comparison with the other examined autophagic mutants
which carry the same apoptosis deficient background. Therefore, the used atg-18(gk378),
atg-7(tm2976) and lgg-1(tm3489) mutations could strongly block autophagic activity,
whereas the unc-51(e369) and unc-51(e1189) mutations not, since the genetic background
of the corresponding double mutants was the same [ced-4(n1162)].
atg-18(gk378); ced-4(n2273) double mutants displayed 36% embryonic and 63%
larval lethality, compared to the full penetrant embryonic lethal phenotype of atg18(gk378); ced-4(n1162) double mutant animals (Fig. 15). This can be explained by the fact
that in ced-4(n2273) hypomorphic mutants apoptosis functions, although with a decreased
intensity, whereas ced-4(n1162) is a null mutation which eliminates all apoptotic events
(Yuan and Horvitz, 1992). In comparison with 100% embryonic lethality in atg-18(gk378);
ced-4(n1162) double mutants, atg-18(gk378); ced-3(n717) animals showed only 47 and 47%
embryonic and larval lethality, respectively (Fig. 15). ced-3(n717) is not a null allele (Shaham
et al., 1999), that could be the cause of the observed weaker phenotype.
The viability of bec-1 silenced nematodes was also examined in ced-4 and ced-3
mutant background. bec-1 null mutations (i.e., ok691 and ok700 alleles, Takacs-Vellai et el.,
2005) cause highly penetrant embryonic lethality themselves, therefore it is difficult to
examine their role in development in apoptotic mutant background. Thus, to reduce bec-1
activity in ced-4 and ced-3 mutant background, we used RNAi treatment, which causes a
weak phenocopy of the bec-1(lf) mutation. Simultaneous silencing of bec-1 and mutational
inactivation of ced-4 or ced-3 led to 58% and 49% embryonic lethality, respectively, which is
significantly higher than that of the corresponding single mutant or RNAi-treated nematodes
(Fig. 21). This experiment also supports the results of the previous autophagy-apoptosis
double mutant analysis. The less penetrant embryonic lethal phenotype of both bec-1(RNAi);
ced-4(n2273) and bec-1(RNAi); ced-3(n717) animals, in comparison with those of the
autophagy-apoptosis double mutants, maybe due to the used technique (RNA interference
vs. mutation).
From these data, it can be concluded that although single mutants for autophagy or
apoptosis produce healthy, wild-type-like offsprings, these cellular processes do function
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during embryonic development of C. elegans. Maternal effect of the autophagy genes
further supports the idea about the importance of autophagy during embryogenesis of
nematodes. The penetrance and expressivity of the observed phenotypes depended on
which autophagy gene was inactivated and also on the character of the mutation.
Additionally, the applied technique whether the corresponding gene was silenced by RNAi or
inactivated by a mutation had also an influence on the resulting phenotypes. It was also
important which apoptosis gene was knocked out and whether the used mutation was
hypomorphic or amorphic. At least one of the two mechanisms should be present for normal
embryogenesis.
5.3 Simultaneous inactivation of autophagy and apoptosis causes
severe morphological malformations during embryogenesis
By analyzing the morphology of autophagy-apoptosis defective double mutant
embryos, I observed that all of them displayed similar characteristics, such as defects in body
elongation, appearance of necrotic-like vacuoles, and the presence of extraembryonic cells.
During the embryogenesis of C. elegans, the epidermal cells drive the elongation of
the worm (Priess and Hirsh, 1986). Between 350-390 minutes of development, the ventral
enclosure takes place, which means that ventral epidermal cells migrate toward the ventral
midline to encase the underlying cells in an epithelial monolayer (Williams-Masson et al.,
1997). Between 395-520 minutes, the ventral enclosure is followed by the elongation phase.
During elongation, a wild-type embryo reduces its circumference by a factor of three and
increases in length by a factor of four. Most of the autophagy-apoptosis defective double
mutants did not display the special shape of a lima bean embryo which is a result of the
ventral enclosure. I suggest that this step, which is strongly necessary for the elongation, is
affected. There are epidermal leading cells which drive the ventral enclosure (WilliamsMasson et al., 1997), and maybe these cells are missing or their movement is blocked in the
analyzed worms lacking both autophagy and apoptosis. There are several mutations, such as
those in evl-20, wsp-1, arx-1, arx-2, arx-5 genes (Antoshechkin and Han, 2002; Sawa et al.,
2003), which cause enclosure defects via impaired cell migration. The morphology of
embryos carrying these mutations is similar to that of autophagy-apoptosis defective double
mutants which supports this hypothesis. Nevertheless, in future experiments it will be worth
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to examine the cell lineage of autophagy-apoptosis defective double mutants to see whether
epidermal leader cells are present in these highly deformed embryos.
During embryonic development of C. elegans, the core apoptotic pathway is
activated in 113 cells, whose degrading materials might serve as nutrients for the surviving
cells (Angelo and Van Gilst, 2009). In the absence of apoptotis, autophagic activity might
compensate for the lack of this nutrient supply. It has been recently described that the
function of autophagy genes in C. elegans are necessary for apoptotic cell corpse
degradation in the engulfing cell (Li et al., 2012). When both autophagy and apoptosis are
compromised, starving embryonic cells might undergo cell death, probably by necrosis. The
observed necrotic-like vacuoles in autophagy-apoptosis defective double mutant embryos
support this idea (Figs. 16, 20). A still not identified regulatory link between the three cell
death pathways might exist, and in nematodes lacking both autophagy and apoptosis,
necrosis might be activated as well, like in the mouse (Kunchithapautham et al., 2007).
Kunchithapautham and colleagues described that treatment of mouse photoreceptor cells
with inhibitors of both autophagy and apoptosis leads to increased cell death by necrosis.
The activation of necrosis as a response for simultaneous inhibition of autophagy and
apoptosis could also be examined in future experiments in C. elegans. The expression of clp1 (calpain family 1), a known necrotic gene, could be analyzed in autophagy-apoptosis
defective double mutant embryos. It would also be interesting to create autophagy-necrosis
and apoptosis-necrosis double, and autophagy-apoptosis-necrosis triple mutant animals, and
examine them as well.
The presence of extra embryonic “shed cells” has already been described in ced-3
single mutants (Denning et al., 2012). These are cells that are localized inside the eggshell,
but outside and separately from the soma. They show apoptotic characteristics like rounded
shape and TUNEL positivity, but they die in a caspase-independent manner (Denning et al.,
2012). This could also mean that the embryo lacking the basic cell elimination mechanism
tries to dispose of the superfluous cells.
In the analyzed autophagy-apoptosis double mutant embryos, despite of the
morphological defects different tissues, complete organs seemed to appear. The pharynx
was generated, gut cells started to form, since their special autofluorescent granules
developed, and working muscle cells were present as animals were able to move. Thus
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differentiation of different cell types may not be affected in the absence of both apoptosis
and autophagy, although these embryos were not able to hatch and died inside the eggshell.
5.4 Autophagy and apoptosis are redundantly required for the
embryonic development of C. elegans
Another approach to analyze the relationship between apoptosis and autophagy is
to examine apoptotic activity in autophagy deficient background and vica versa. Thus, I
measured the level of apoptotic activity in autophagic mutants and I found that it was
increased in comparison with the wild type. Both the cell corpse number of lgg-1 mutants
and the amount of TUNEL positive structures in atg-18 and unc-51 deficient nematodes were
elevated. This indicates that the lack of autophagy activates apoptosis in certain cells.
I found two possible explanations for these results: 1) autophagy acts in certain cells
to protect them from undergoing apoptosis, or 2) in the absence of autophagy the
engulfment process is impaired. The first explanation is supported by the fact that
autophagy is basically a cytoprotective mechanism. It might rescue cells from apoptosis in
wild-type animals, but in the absence of autophagy the apoptotic machinery is able to
eliminate these cells. It is still an open question whether this effect is conserved in the C.
elegans cell lineage, i.e. depletion of autophagy allows the death of the same cells in every
animal.
It is already known that autophagy participates in the clearance of apoptotic cells (Li
et al., 2012). According to the second model, the apoptotic activity is not elevated, but the
corpses are present in the embryos for a longer period of time. To decide which explanation
is correct, the cell lineage of autophagic mutants should be mapped, and it should be
determined which cells die during embryonic development when autophagy is absent. Thus,
dead cells could be identified whether they overlap with the group of cells that are normally
destined to die by apoptosis in wild-type animals, or they are resulting from additional
apoptotic events.
I also examined autophagic activity in apoptosis defective mutant background,
compared to the wild type by visualizing the accumulation of GFP::LGG-1 fusion protein, a
wildly used marker of autophagy. Since ced-4 is highly active and most of the apoptotic
events occur during embryogenesis, I expected changes in the expression pattern of lgg-1 in
83
ced-4 mutant embryos. Unexpectedly, I could observe no difference between gfp::lgg-1 and
gfp::lgg-1; ced-4(n1162) strains.
LGG-1 is present in almost all embryonic cells with a basal activity, and this is
considerably high during early development. Additionally, transgenic animals used in our
study contain high copy number of the marker construction. Furthermore, only 1-2 apoptotic
events can be observed at a given time point and I expected elevated expression of the
marker gene only in these cells. Considering these facts, it is possible that the used system is
not sensitive enough to detect this relatively rare, punctuated change in the expression.
The synthetic lethal phenotype of autophagy-apoptosis defective double knockouts
and the fact that apoptotic activity is elevated in autophagic mutant background suggest
that the two mechanisms act redundantly during embryonic development of this model
organism. The entire cell lineage of C. elegans is mapped and in a wild-type hermaphrodite
131 cells, whereas in a wild-type male 147 cells die during development (Sulston and Horvitz,
1977; Sulston et al., 1983). With the exception of the male-specific linker cell death, all of
these cells die by apoptosis (Blum et al., 2008). Under normal conditions autophagic cell
death has not yet been described during the embryogenesis of C. elegans. Although it does
not exclude the possibility that ACD is present under certain environmental circumstances.
In summary, the two mechanisms are essential together for the normal development of C.
elegans, although our experiments do not reveal whether autophagy acts as a cytoprotective
process, as a cell death mechanism or both during development.
5.5 Autophagy and apoptosis together have an influence on the
fertility of C. elegans
Silencing of bec-1 by RNAi in ced-4 and ced-3 mutant background resulted in
increased embryonic lethality and sterility compared to the corresponding single mutants
and RNAi silenced animals. bec-1(RNAi); ced-3(n717) animals displayed a similar phenotype
as found in bec-1(RNAi); ced-4(n2273) animals (Fig. 21).
Next, I also measured the brood size of autophagy-apoptosis defective double
mutants compared to the corresponding single mutants. I chose atg-18-apoptosis defective
double mutants for this experiment since atg-18 is the only autophagy-specific C. elegans
gene, and atg-18(gk378) is the strain which was crossed with a series of apoptosis mutants. I
84
measured the progeny number of the F1 generation, since the F2 generation displayed full
penetrant embryonic lethality.
atg-18(gk378); ced-4(n1162), atg-18(gk378); ced-4(n2273) and atg-18(gk378); ced3(n717) double mutants showed a synthetic brood size reduction phenotype, as compared
to the corresponding single mutants (Fig. 22). The brood size of a C. elegans specimen
depends on the number of gametes, but mostly on the amount of the sperms produced.
However defects in either oogenesis or spermatogenesis can lead to a brood size reduction
phenotype. It has been described that superfluous oocytes are eliminated from the C.
elegans germline via the core apoptotic pathway (Gumienny et al., 1999). On the other
hand, there has not been observed any apoptotic activity during the generation of sperm
cells (Gumienny et al., 1999). Although there is no evidence about the autophagic function in
the germline of C. elegans, the role of this cellular mechanism in oogenesis of other model
organisms is known (Barth et al., 2011; Gawriluk et al., 2011). Thus the function of
autophagy and apoptosis may be present rather in oogenesis than spermatogenesis.
According to an existing model, certain germ cells in the pachytene phase serve as
nurse cells for maturing oocytes. The cytoplasmic volume of the apoptotic nurse cell is a
supply which is essential for growth of developing oocytes. In the absence of apoptosis
maybe autophagy serves as a backup mechanism to ensure nutrient supply for oocytes.
5.6 The role of bec-1 in the regulation of autophagy and apoptosis
The role of bec-1 in the mechanism of autophagy is intensively studied. It is
described that bec-1/BECN1 also participates in apoptosis, however its exact role in this type
of PCD remains to be established. Human beclin 1 (BECN1) and its nematode counterpart
BEC-1 were shown to interact with the anti-apoptotic proteins Bcl-2 and CED-9, respectively
(Pattingre et al., 2005; Takács-Vellai et al., 2005). However, unlike other known BH3-only
proteins, Beclin 1 does not function as a pro-apoptotic molecule, even if it is overexpressed
(Kang et al., 2011). The interaction of Beclin 1 and Bcl-2 does not result in losing the antiapoptotic potential of Bcl-2 (Ciechomska et al., 2009). Furthermore, several circumstances,
like irradiation, nutrient deprivation or hypoxia, enhance the anti-apoptotic function of
Beclin 1. In C. elegans and also in mammalian cell cultures, depletion of bec-1/BECN-1
triggers caspase-dependent programmed cell death (Takács-Vellai et al., 2005; Boya et al.,
2005). The precise mechanism by which Beclin 1 inhibits apoptosis is not yet clear, but
85
maybe related to disregulated autophagy. Interestingly, caspases can cleave Beclin 1 in
apoptosis, thereby destroying its pro-autophagic activity (Djavaheri-Mergni al., 2010). Thus,
BEC-1 might have an essential role in integrating and fine-tuning autophagic and apoptotic
signals.
Based on the autophagy-apoptosis double mutant analysis, the two mechanisms
function redundantly during embryonic development. Therefore we assumed a shared
regulation of autophagy and apoptosis. This prompted us to search for potential binding
sites of transcription factors that are known modulators of apoptotic genes in the regulatory
regions of autophagy genes. In this manner we found a sequence motif in the promoter of
bec-1 which differs in only one base pair from the consensus binding site of CES-2 and ATF-2
bZip-like transcription factors. This element is conserved among several Caenorhabditis
species (Fig. 26), thus the regulation of bec-1 and its orthologs through this sequence are
phylogenetically conserved.
Next, we proved that both CES-2 and ATF-2 physically interact with this region of
the bec-1 promoter in vitro. In the gel retardation assay neither of the two transcription
factors was shown to interact with the binding site when it was mutated, thus the proteinDNA interactions take place specifically (Fig. 27).
Finally, we confirmed our in vitro results in an in vivo assay. The expression level of
the mutated bec-1::gfp reporter construct, in which the potential bZip-like binding site was
deleted, was elevated in several tissues in embryos, as well as in L1 and L4 larvae as well, in
comparison with that of the wild-type construct (Figs. 29, 30, 31). In embryos, bec-1 can be
modulated either by ATF-2 or CES-2, or maybe by both of them, since they are expressed in
this developmental stage and it is known that the bZip-like transcription factors can function
as homodimers and heterodimers as well. In larvae, only ATF-2 is expressed (Wang et al.,
2006). Thus, in postembryonic developmental stages this bZip-like transcription factor
should regulate bec-1. My colleague, Éva Borsos investigated the effect of ATF-2 and CES-2
on the regulation of an other autophagy gene, lgg-1 (Erdelyi et al., 2011). lgg-1 also contains
a bZip-like binding site in its promoter. She demonstrated that explession levels of gfp::lgg-1
are increased in atf-2 loss-of-function mutant embryos. In that case, not only the binding site
was proven to be functional, but ATF-2 was indeed able to repress lgg-1. Together, these
data suggest that the autophagy genes lgg-1 and bec-1 are negatively regulated by the bZiplike transcription factor ATF-2, and maybe by CES-2 as well.
86
I set up a model where the ATF-2(CES-2?)/bec-1 interaction contributes to cell fate
decision. In some cells that are fated to live the amounts of ATF-2 and CES-2 are low, thus
bec-1 can be expressed to support autophagy. Next, autophagy as a cytoprotective
mechanism antagonizes apoptosis, thereby enabling the affected cells to survive. The
question whether bec-1 directly blocks apoptosis or this inhibition is a result of the induction
of autophagy, needs to be further investigated (Fig. 32). On the other hand, in cells destined
to die the level of bZip-like transcription factors is high, resulting in the inhibition of
autophagy and the promotion of apoptosis via the repression of bec-1 (Fig. 32).
Figure 32. Schematic illustration of the model about the regulation of autophagy and apoptosis via
the repression of bec-1 by ATF-2 – According to this model, the level of ATF-2, and/or maybe CES-
2, is low in those cells of the embryos which are fated to live. Here, bec-1 can be expressed,
supporting autophagy and inhibiting apoptosis. In cells destined to die, high amount of ATF2, and/or maybe CES-2, blocks bec-1 which results in low levels of autophagic activity and
the induction of apoptosis.
These new data about autophagy and apoptosis can lead to elucidate the functions
of these highly important biological mechanisms. We suggest that autophagy and apoptosis
are redundantly required for viability, and partially for fertility in C. elegans. Furthermore,
we showed that these processes share a common upstream transcriptional control by the
bZip-like transcription factors ATF-2 and CES-2. These results can help to understand the
interplay between autophagy and apoptosis during development.
87
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106
Chapter II.
A functional analysis of a newly identified activation loop of myosin
in C. elegans
107
7 General introduction
At the Department of Biochemistry, Eötvös Lorand University, András MálnásiCsizmadia and colleagues work on actin-myosin interaction and muscle force generation. In
collaboration with them, together with Boglárka Várkuti we set up an in vivo model system
to analyse the function of an activation loop of myosin in the actomyosin system.
7.1 The muscle structure of C. elegans
Two main types of muscle exist in C. elegans: obliquely striated (also called somatic
muscle or multiple sarcomere) muscle and nonstriated (also called single sarcomere) one. In
hermaphrodites, the 95 body wall muscle cells belong to the multiple sarcomere group. The
single sarcomere group consists of 20 pharyngeal muscle cells, 2 stomato-intestinal muscle
cells, 1 anal sphincter muscle cell, 1 anal depressor muscle cell, 8 vulval muscle cells, 8
uterine muscle cells and the contractile gonadal sheath cell. In males, instead of the vulval
and uterine muscles and gonadal sheath, 41 specialized mating muscle cells are present,
some having single sarcomeres and some being obliquely striated (Hall and Altun, 2008).
In muscle cells, the basic unit of the contractile apparatus is the sarcomere. In the
body wall muscle repeats of the sarcomere give the "striated" appearance. In vertebrates, a
sarcomere containes two Z (Zwischenscheibe) discs located at each end of a sarcomere, two
halves of I (Isotropic) bands, corresponding to thin filaments, an A (Anisotropic) band,
corresponding to thick filaments, an H (Heller) zone, corresponding to the central region of
the A band, and an M (Mittel) line at the middle of the H zone where each myosin rod is
joined with its neighbor. In the sarcomere, myosin-containing thick filaments and actincontaining thin filaments are interdigitated with each other. In C. elegans, dense body (DB) is
analogous to the Z disc in vertebrates (Fig. 33). Its function is to anchor and align thin
filaments in striated muscles. Thick filaments are attached to M line analogs. Both the DB
and the M line analogs anchor filaments to the cell membrane, the underlying hypodermis,
and cuticle (Waterston, 1988).
In C. elegans, the 95 body wall muscle cells are arranged along four “zigzag” lines
begetting four longitudinal quadrants. Three of these quadrants contain 24 cells, whereas
the fourth quadrant contains only 23 cells. A thin basal lamina (BL) is located between the
muscles, and the underlying hypodermis, and nervous tissue. Somatic muscle cells can be
108
divided in three parts: the contractile filament lattice, a noncontractile body containing the
nucleus and the cytoplasm with mitochondria, and the muscle arms that extend to either
ventral or dorsal nerve cords or the nerve ring.
Figure 33. Somatic muscle structure of C.elegans – Myosisns (yellow lines) are attached to the M
lines and actins (black lines) are linked to the Z disc analog dens bodies. The rows of M lines and
dense bodies occur to deflect with an angle of 5–7° from the longitudinal axes of the filaments
(based on Hall and Altun, 2008).
Although the filaments are oriented parallel to the longitudinal axis of the muscle
cell, neighboring structural units, such as M lines and DBs, are offset from each (Waterston,
1988; Bird and Bird, 1991). Therefore, A–I striations occur to deflect with an angle of 5–7°
from the longitudinal axes of the filaments and the muscle cell (Fig. 33). On the contrary, this
angle is 90° in the vertebrate cross-striated muscles. It is suggested that this obligate
arrangement of the sarcomeres is advantageous since the generated muscle force is more
evenly distributed over the BL and cuticle, resulting in smooth bending of the body rather
than kinking (Burr and Gans, 1998).
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7.2 The myosin
Myosins are molecular motor proteins that convert chemical energy (ATP) to
mechanical energy, that resulting in generation of force and movement. Myosins constitute
a diverse protein family (currently grouped at least into 35 classes) and are ubiquitously
present in all eukaryotic cells (Odronitz et al., 2007). Beside muscle contraction, cellular
motor functions - including cell proliferation and differentiation - and intracellular transport
are also powered by different types of myosins (Pollard, 2000).
Myosin II participates in the force generation of muscles. This is a very large protein
(about 500 kDa) which consists of two identical heavy chains (about 200 kDa each) and two
pairs of light chains (about 20 kDa each). Heavy chains contain a globular head region, a neck
and a long α-helical tail. The tails of the two heavy chains twist around each other and form
a coiled-coil structure. The two pairs of light chains associate to the neck regions (Cooper,
2000).
The head is the motor domain which binds actin and hydrolyses ATP to generate
force. The neck domain functions as a linker and as a lever arm for transducing the
generated force. Another role of the neck domain is to serve as a binding site for distinct
myosin light chains which generally have regulatory functions. The tail domain interacts with
cargo molecules and/or other myosin subunits. Furthermore, it plays a role in regulating
motor activity (Sweeney and Houdusse, 2010).
Several hundred myosin molecules connected to each other by their tails to form a
thick filament. Their heads are arranged in a staggered layout along the bundle of the tails.
Thin filaments are made up of several globular actin monomers with identical orientation,
giving polarity to the filament. Myosin binds actin by the globular head, forming crossbridges between the thick and thin filaments. The tails of myosin molecules in the thick
filaments are oriented towards the M line in both halves of the sarcomere. Actin filaments
attach to Z discs at their plus ends. Their polarity is similarly reverses at the M line. Thus, the
relative orientation of thick and thin filaments is the same. During muscle contraction, the
myosin head groups move along the actin filaments in the direction of the Z disc since
myosin II is a plus-end motor protein. Therefore actin filaments slide from both sides of the
sarcomere toward the M line. In this way, the muscle contracts due to the shortening of the
sarcomeres (Fig. 34).
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Figure 34. Sliding model of contraction – Myosin filaments are located among actin filaments. Upon
contraction, the heads of the myosins move along actin filaments in the direction of the + ends, thus
finally the sarcomeres shorten (based on Cooper, 2000).
Myosins act in complex with actin I and II. Despite the large functional variety of
myosins, there is a universal molecular mechanism underlying the acto-myosin
chemomechanical cycle. The cyclic actin binding and release alternating with the swing and
structural relaxation of the myosin lever define the mechanical cycle of actomyosin that is
coupled with the chemical cycle, including the nucleotide binding, hydrolysis and product
release steps. Actin accelerates the steady-state ATPase activity of myosin, which is known
as actin activation, by increasing the rate constant of the lever swing, thereby channeling the
enzymatic system toward the effective chemomechanical pathways (Málnási-Csizmadia and
Kovacs, 2010). The molecular mechanism behind actin activation of myosin that determines
the force and velocity of muscle contraction is not yet known.
In C. elegans, four different myosin II coding genes exist: myo-1, myo-2, myo-3 and
myo-4. myo-1 and myo-2 are expressed only in the muscle of the pharynx. myo-3, encoding a
minor isoform that is essential for thick filament formation (Waterston et al., 1989), and
myo-4 are expressed primarily in the body-wall muscle. myo-4, also called unc-54, is the
major component of the force generation in the body-wall muscle thus essential for the
movement of the nematode (Moerman et al., 1982).
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8 Aims
Preliminary data indicated that a newly identified loop of myosin is essential for
actin activation. My PhD student college, Boglarka Varkuti, proved by both in silico and in
vitro assays that the positively charged Arg520 in the activation loop of myosin motor
domain forms a salt bridge cluster with the negatively charged N-terminal peptide of actin.
This interaction participates in actin activation and in the effective force generation.
We intended to support these results with the analysis of this myosin loop in an in
vivo model. According to this, we planned to rescue a myosin null mutant C. elegans
strain,unc-54(e1092), with a DNA construct containing the promoter and the full length ORF
of unc-54, and also with the altereded version of this construct mutant for the coding region
of the activation loop. In both constructs the unc-54 gene was tagged with the sequence of a
gfp marker.
We planned to compare the expression pattern of the transgenic lines carrying
either the wild-type or the mutated transgene integrated into the genome in low copy
number. Next, we intended to compare their speed, progeny number and lifespan in order
to conclude the importance of the activation loop. Finally, we wished to exclude the
possibility that the differences between the transgenic lines were due to the differences in
the expression level of the integrated transgene. To see this we measured whether the
levels of expression correlated with the speed of the worms
112
9 Matherials and Methods
For maintenance and cleaning of C. elegans strains, generation of transgenic
animals (microparticle bombardment) and use of light and fluorescence microscopy see
Methods in Chapter I.
9.1 C. elegans motility assay
All recordings were taken with a Zeiss Lumar.V12 microscope and with Zeiss
AxioCam using AxioVision 4.8 software. All videos were analyzed by the δVision software.
For determining the speed, videos were taken and analyzed from 40-40 animals from each
strain. For measuring the velocity of the animals during their lifetime, first 20-20 worms from
each strain were transferred onto separated NGM plates supplemented with 300 mg/ml
FUdR to sterilize them. All of them were recorded every day until they got paralyzed. Finally,
the videos were analyzed and the data were summarized for each day for each strain. For
calculation of the correlation between the speed and the expression level videos and
fluorescent images were taken from 40-40 worms from both transgenic lines individually.
Relative expression levels were calculated by measuring the average pixel intensity of the
worms in the fluorescent images. Later the measured speed and the relative expression level
were compared.
9.2 Determining brood size and the number of bags of worms
For determining the brood size, 10 adult worms were put on separated plates. All
the animals were transferred on a new plate every day, and the layed eggs from the previous
24 hours were counted on each plate. This was repeated until the animals became fertile.
To measure the rate of bag of worms, 10-10 young adults were transferred onto
new NGM plates from each strain. Every day the number or bag of worms was counted and
the survivors were put onto new plates.
9.3 Lifespan assay of C. elegans
All assays were carried out at 25°C with synchronized C. elegans cultures. For the
lifespan assay L4 animals were transferred to NGM plates supplemented with 300 mg/ml
FUdR. Surviving animals were counted each day. Animals were considered dead when they
113
stopped pumping with their pharynx and responding to touch. Lifespan statistics were
calculated by Keplen-Meier analysis (SPSS software).
9.4 Used and generated strains
N2 wild type strain
CB1092 unc-54(e1092)I.
BU292 unc-54(e1092)I.; pMA001[int.punc-54::unc-54::GFP]
BU293 unc-54(e1092)I.; pMA002[int.punc-54::unc-54(K525E)::GFP]
114
10 Results
Preliminary results indicated that a newly identified activation loop in the myosin
protein is required for actin activation and hereby essential for the effective muscle
contraction. To analyze this module of myosin in an in vivo system, we tried to rescue the
paralyzed phenotype of the unc-54(e1092) mutants with a construct containing 3.5 kbps
promoter region and the full length ORF of unc-54, tagged with the sequence of gfp marker
in the 3’ end (called unc-54::gfp). We carried out the rescue also with an altered version of
this construct containing an A to G substitution mutation in the sequence of the activation
loop (called unc-54K525E::gfp). This resulted in the changing of the 525th amino acid of
myosin which is a basic lysine (K) to an acidic glutamate (E). unc-54(e1092) is a null mutation
carrying a C/T substitution resulting in the appearence of a premature ochre stop codon
(Dibb et al., 1985). C. elegans strain homozygous for this allele does not express myosin
heavy chain B (MHC-B) (Anderson and Brenner, 1984).
Both unc-54(e1092); unc-54::gfp and unc-54(e1092); unc-54K525E::gfp transgenic
animals showed the same expression pattern: the 95 body wall muscle cells, the vulval- and
anal depressor muscles were labeled. In the muscle cells the GFP markered double lines
indicate a series of A bands (Fig. 35).
a
b
c
d
e
f
g
h
i
j
Figure 35. Expression pattern of the unc-54(e1092); unc-54::gfp and unc-54(e1092); unc-54K525E::gfp
transgenic animals – The wild-type unc-54::gfp construct is expressed in the whole body-wall musce
(a, b, c), so as is the mutated version (f, g, h). The vulval muscles are also labeled by both unc-54::gfp
(d) and unc-54K525E::gfp (i). The romboid shape of the muscle cells can be observed in both strains
(dotted lines in fig. e – wild type construct, and in fig. j – mutated construct).
115
After the generation and the expression pattern analysis of transgenic strains we
measured their speed in comparison with the wild type and the unc-54(e1092) single
mutants to see whether the rescue was successful. unc-54(-) mutants could move only with
17% of the wild-type speed. Whereas transgene containing wild-type unc-54 could raise the
speed of the mutants up to 70% of a wild-type movement, the mutated transgene could only
double the speed of the paralyzed unc-54 mutants (Table 11, Fig. 36). Thus, we could
partially rescue the uncoordinated phenotype and the analyzed loop seems to be essential.
Table 11. Speed of the wild-type animal, unc-54 mutant, unc-54 mutant that are rescued by the
wild-type construct, and unc-54 mutant that are rescued by the mutated construct.
genotype
speed (μm/s)
speed compared to
N2 (%)
232.30
100.00
38.82
16.71
164.84
70.96
82.50
35.51
N2
unc-54(e1092)
unc-54(e1092); unc-54::gfp
unc-54(e1092); unc-54(K525E)::gfp
Speed (μm/s)
300
250
N2
200
unc-54(e1092)
150
unc-54(e1092);
unc-54::gfp
100
unc-54(e1092);
unc-54(K525E)::gfp
50
0
Figure 36. Speed of the wild type, unc-54 mutants, unc-54 mutants which are rescued by the wild
type construct and unc-54 mutants which are rescued by the mutated construct – The speed of
wild-type C. elegans was about 230 μm/s. Mutational inactivation of the unc-54 gene reduced the
speed of the animals to one sixth. This was partially rescued when a construct, containing a gfptagged wild-type unc-54 gene, was inserted into the genome of these mutants. When a specifically
mutated version of this construct was inserted into the genome of the unc-54 mutants, the effect of
the rescue was significantly reduced.
116
Since unc-54 is expressed in the vulva muscles, worms carrying the e1092 allele
display an egg-laying defective phenotype, which means the accumulation of embryos in the
mother due to the reduction of egg-laying efficiency. In worms highly unable to lay eggs,
embryos finally hatched caused the death of the mother. The phenotype when the larvae
are inside their mother is called „bag of worms”. We measured whether the two transgenes
could rescue the egg-laying phenotype as well. 90% of unc-54(e1092) single mutants became
bag of worms, while 100% of wild-type animals could lay all of their produced embryos. The
ratio of the bag of worms in both wild-type and mutant transgenic strains were 40%. The
produced progeny number of the unc-54(e1092) single mutants was onlyquarter as
compared to those in the wild type. Both transgenes could double the progeny number of
the mutants, but there were no significant difference between them (Fig. 37).
100
90
Progeny number
80
N2
70
unc-54(e1092)
60
50
unc-54(e1092);
unc-54::gfp
40
30
unc-54(e1092);
unc-54(K525E)::gfp
20
10
0
1
2
3
4
5
6
Days
Figure 37. The progeny number of the analyzed strains during the reproductive phase of their life –
The rescue of both wild-type and mutated construct was only partial. There was no significant
difference between the two rescuing constructs. Additionally, the pattern of all curves is similar.
To avoid the influence of the carried eggs on the movement (unlayed eggs were
accumulated in the mothers, thus the additional weight and the wider body detained them),
we
treated
the
animals
with
FudR
(5-Fluoro-1-[4-hydroxy-5-(hydroxymethyl)
tetrahydrofuran-2-yl]-1H-pyrimidine-2,4-dione). This inhibits cell proliferations, thus the
treated animals did not hold any embryos due to their sterility. Additionally, this resulted in
the survival of animals which would otherwise became bag of worms, thus we could
measure a group of samples which was more representative to the population. We checked
117
the movement of all animals every day until they got paralyzed (few days before their death
worms become paralyzed independently from their genetic background). With this setting of
the experiment we measured the same difference between the wild type and unc-54(e1092)
single mutant animals, like without FudR treatment; the speed of the mutants was reduced
to 20% of those in the wild type. Interestingly, the transgene containing the wild-type unc-54
gene could fully rescue the paralyzed phenotype caused by the e1092 allele. unc-54(e1092);
unc-54K525E::gfp transgenic animals displayed only a slightly increased speed compared to the
unc-54(e1092) animals (Fig. 38). The pattern of lifetime speed curve of all measured strains
was similar. Animals showed the highest velocity at the first day of adulthood, then, during
the next 2-3 days, they could move with a slightly reduced but still a relatively high speed
which was constant. Finally, their movement became significantly slower and slower from
day to day untill they got paralyzed. Interestingly, the start of the decay in movement at
about the 4th day of their adulthood roughly coincides with the end of the reproductive
phase of their life (Fig. 38).
300
N2
Speed (μm/s)
250
200
unc-54(e1092)
150
unc-54(e1092);
unc-54::gfp
100
unc-54(e1092);
unc54(K525E)::gfp
50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days
Figure 38. The velocity of the FUdR-treated strains during their lifetime – After FUdR treatment, the
animals became sterile, thus the disorder in the egg-laying did not influence speed. Under these
circumstances, the rescue by the wild-type construct was complete in the first four days. In contrast
with this, the speed of unc-54 mutants carrying the mutated construct were only slightly higher in
comparison with those of the unc-54 mutants.
118
Next, we analyzed whether the speed of the transgenic animals correlated with the
expression of the transgenes. We compared the expression level and the velocity of a series
of animals carrying either the wild-type or the mutant construct. Although the average
expression level of the mutated construct was lower than that of the wild-type construct,
there was no correlation between gfp exprerssion and velocity of the animals (Table 22, Fig.
39).
Table 12. The correlation of the speed and expression level of the transgenic strains
genotype
speed (μm/s)
unc-54(e1092);
unc-54::gfp
unc-54(e1092);
unc-54(K525E)::gfp
pixel intensity
speed/Pixel
intensity
pearson's
correlation
164.84±49.5
34.74±8.75
4.88 ± 1.35
0.29052
82.5±25.5
21.65±2.99
3.88 ± 1.53
0.20638
Average pixelintensity
80
70
unc-54(e1092); unc54::gfp
60
unc-54(e1092); unc54(K525E)::gfp
50
40
Linear (unc-54(e1092);
unc-54::gfp)
30
20
Linear (unc-54(e1092);
unc-54(K525E)::gfp)
10
0
0
100
200
300
400
Speed (μm/s)
Figure 39. The correlation of the speed and expression level of the transgenic strains – The
deviation of both the speed and the gfp expression level of worms carrying the wild-type construct
was higher, compared to those containing the mutated construct. However, there was no correlation
between these values in either strain.
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Figure 40. Lifespan curves of the four analyzed strains – The average lifetime of the unc-54(e1092)
mutants was reduced, as compared to the wild type. Integration of either the wild-type or the
mutated unc-54 construct into the genome of these mutants coud not rescue the shortened lifespan
phenotype.
C. elegans is a highly suitable modell organism for lifespan analysis since it is a
sensitive system for both the exterior and the interior effects. Thus, we measured the
lifespan of transgenic animals, compared to the wild type and the unc-54(e1092) single
mutants, in order to see whether the transgenes caused any unexpected defects. The
lifetime of unc-54(e1092) mutants was two days shorter than that of the wild type, which
was 13 days (Fig. 40). This difference is significant based on Kaplan-Meier analysis. Both unc54(e1092); unc-54::gfp and unc-54(e1092); unc-54K525E::gfp transgenic lines displayed a
similar lifespan as the unc-54(e1092) mutants (Fig. 40). Thus, none of the transgenes were
able to rescue the reduced lifespan of the single mutant and there was no significant
difference between the lifetime of animals containing the wild-type or the activation-loopspecific mutant construct. The integration and expression of the transgenes did not affect
the lifespan.
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11 Discussion
The major aim of our experiments was to examine in an in vivo system whether the
newly identified activation loop of myosin is functional. Animals defective for unc-54 are
paralyzed, thus the major isoform of body wall musle myosin is essential for effective force
generation and movement (Anderson and Brenner, 1984). This provides a good opportunity
to investigate the function of myosin. According to this, we set up a system in which unc-54
mutant nematodes lacking body wall muscle myosin was rescued by constructs containing
either a wild-type unc-54 gene or its mutated version where a substitution was carried out in
the sequence of the activation loop. The mutation resulted in the change of a basic lysine,
the key amino acid of the activation loop, to an acidic glutamate. By microparticle
bombarding system, low copy number homozygous integrated lines were generated using
both constructs.
The expression pattern of both transgenes was almost identical: it was expressed in
the body wall musce cells, in the vulval muscles and in the anal depressor muscles. It was
identical with that described previously in the unc-54 gene (Fire and Waterson, 1989). In
animals containing the wild-type unc-54, the expression level of the gfp was slightly higher
than that in worms carrying unc-54K525E. This difference maybe a result of the difference in
the copy number of the integrated transgenes or could be due to the location of the
integrations in the genome. This difference in the expression levels did not correlate with
the speed of the worms, i.e., the rate of the rescue. The morphology and development of
the transgenic animals were superficially similar to those of the wild type. Also the lifespan
of both of them did not differ significantly from that of the unc-54(e1092) mutants. Thus, the
integrated transgenes seems not to cause any defect in the phisiology of the transgenic
animals. Alltogether, these data indicate that the newly generated strains can be used for
investigation of the functionality of the activation loop of myosin.
The contruct containing the wild-type unc-54 gene partially rescued the paralyzed
phenotype of unc-54 mutants. However, when these transgenic animals were sterile due to
the FUdR treatment, the rescue was complete. This maybe a result of the difference in
carried embryos, since the rescue of the egg-laying phenotype of unc-54 single mutants was
incomplete. Compared to the rescue caused by the wild-type construct, unc-54K525E::gfp
transgene was only able to slightly increase the motility of unc-54 mutants in both cases.
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This increase in speed of the activation loop deficient animals compared to the null mutants
could be a result of the relatively high number of myosin molecules in muscle cells. It has
been shown in the paper of Boglarka Varkuti by transmission electron microscopy images,
that transgenic worms expressing either unc-54::gfp or unc-54K525E::gfp showed intact fiber
and obliquely striated sarcomere structures of the body-wall muscles along the entire body.
By contrast, unc-54 mutant worms had a stunted, disorganized sarcomere structure because
they lack the major MYO-4 isoform (Varkuti et al., 2012).
All these data indicate that the basic lysine of the newly identified activation loop is
functional. It is necessary for the effective force generation thereby the muscle contraction.
Since this loop seems to be present in all types of myosins independently of function, this
new piece of knowledge could lead to better understanding of how the muscles and all other
systems, in which the acto-myosin system is involved, work.
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12 References
Anderson P, Brenner S (1984). A selection for myosin heavy chain mutants in the nematode
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 81, 4470–4474.
Bird AF, Bird J (1991). The structure of nematodes. Academic Press, San Diego, CA.
Burr AHJ, Gans C (1998). Mechanical significance of obliquely striated architecture in
nematode muscle. Biol. Bull. 194: 1-6.
Cooper GM (2000). The Cell, 2nd edition A Molecular Approach Ch11-Actin, Myosin, and Cell
Movement.
Dibb NJ, Brown DM, Karn J, Moerman DG, Bolten SL, Waterston RH (1985). Sequence
analysis of mutations that affect the synthesis, assembly and enzymatic activity of
the unc-54 myosin heavy chain of Caenorhabditis elegans. J. Mol. Biol. 183(4):54351.
Fire A, Waterston RH (1989). Proper expression of myosin genes in transgenic nematodes.
EMBO J. 8(11):3419-28.
Hall DH, Altun ZF (2008). C. elegans atlas, Ch5(145)
Málnási-Csizmadia A, Kovacs M (2010). Emerging complex pathways of the actomyosin
powerstroke. Trends Biochem. Sci. 35, 684–690.
Moerman DG, Plurad S, Waterston RH, Baillie DL (1982). Mutations in the unc-54 myosin
heavy chain gene of Caenorhabditis elegans that alter contractility but not muscle
structure. Cell 29, 773–781.
Odronitz F, Kollmar M (2007). Drawing the tree of eukaryotic life based on the analysis of
2,269 manually annotated myosins from 328 species. Genome Biol. 8, R196.
Pollard TD (2000). Reflections on a quarter century of research on contractile systems.
Trends Biochem. Sci. 25, 607–611.
Sulston JE, Horvitz HR (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis
elegans. Dev. Biol. 56(1), 110-56.
Sweeney HL, Houdusse A (2010). Structural and functional insights into the Myosin motor
mechanism. Annu. Rev. Biophys. 39, 539–557.
Várkuti BH, Yang Z, Kintses B, Erdélyi P, Bárdos-Nagy I, Kovács AL, Hári P, Kellermayer M,
Vellai T, Málnási-Csizmadia A (2012). A novel actin binding site of myosin required
for effective muscle contraction. Nat. Struct. Mol. Biol. 19(3):299-306.
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Waterston RH (1989). The minor myosin heavy chain, mhcA, of Caenorhabditis elegans is
necessary for the initiation of thick filament assembly. EMBO J. 8, 3429–3436.
Waterston RH (1988). Muscle. In "The nematode C. elegans" Cold Spring Harbor Laboratory
Press, New York. pp281-335.
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13 Summary
Programmed cell death (PCD) plays a fundamental role in animal development and
tissue homeostasis. There are three types of PCD: apoptosis (type I PCD), autophagy (type II
PCD) and necrosis (type III PCD). Apoptosis is the most studied and best understood form of
PCD. After the initiation of this type of cell death functional proteins are cleaved by caspases,
the DNA is fragmented and finally the cell is engulfed by specific or neighbouring cells.
Autophagy is a highly regulated self-degradation process in eukaryotic cells. It functions in
cellular homeostasis as well as it is a cytoprotective response mechanism during
environmental stresses. On the other hand, excessive autophagy can lead to cell death
under certain circumstances. In my work I focused on the interplay between apoptosis and
autophagy in the modell system Caenorhabditis elegans.
Interestingly, deficiency in either apoptosis or autophagy does not affect the viability
and fertility of C. elegans. In contrast with these, inactivation of autophagy genes in
apoptotic mutant background caused embryonic or early larval lethality. These apoptosisautophagy double mutant embryos showed serious morphological malformations.
Furthermore, we observed a significantly increased level of apoptosis in autophagy deficient
background compared to wild type. Interestingly, the expression of the gfp::lgg-1 autophagic
marker construct did not show any change in apoptotic mutant background in comparison
with wild type. Simultaneous inactivation of these two mechanisms negatively affected the
fertility of the nematodes as well. These results indicate that the apoptotic and autophagic
pathways are redundantly required for the viability and development of C. elegans.
Next we searched for common regulators of autophagy and apoptosis. A potential
binding site of the bZIP-like CES-2/ATF-2 apoptosis related transcription factors was found in
the upstream regulatory sequence of the bec-1 gene. This site was conserved among
Caenorhabditis species. By in vitro gel shift assay, we detected efficient and specific binding
of both CES-2 and ATF-2 to the bec-1 promoter. Furthermore, in an in vivo experiment
bZip∆_bec-1::gfp displayed ectopic accumulation in certain cell types, as compared to the
wild-type construct. These results suggest that ATF-2 and CES-2 repress the activity of the
bec-1 autophagy gene, and this regulation may contribute to the cell-fate decision during
development.
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14 Összefoglalás
A programozott sejthalál (PCD) alapvető fontosságú az egyedfejlődésben és a
szövetek homeosztázisában. A PCD-nek három különböző típusát különböztetjük meg: az
apoptózist (I-es típusú PCD), az autofágiát (II-es típusú PCD) és a nekrózist (III-as típusú PCD).
Az apoptózis a PCD leginkább tanulmányozott és a legismertebb fajtája. A folyamat
iniciációja során a fehérje bontó kaszpázok aktiválódnak, majd a DNS fragmentálódik és
végül az elhalt sejtet erre specializálódott sejtek vagy a szomszédos sejtek bekebelezik. Az
autofágia az eukarióta sejtek szabályozott önemésztő folyamata. Szerepe van a sejtek
homeosztázisának fenntartásában, valamint véd a különböző környezeti stresszhatásokkal
szemben. Bizonyos körülmények között azonban a túlzott autofág aktivitás sejthalálhoz
vezethet. Munkám során az autofágia és az apoptózist közötti kölcsönhatást vizsgáltam a
Caenorhabditis elegans modell rendszeren.
Érdekes módon, sem az apoptózis sem az autofág aktivitás hiánya nem befolyásolja a
C. elegans életképességét illetve termékenységét. Bemutattuk, hogy ezzel ellentétben az
autofág gének inaktiválása apoptózis mutáns háttéren embrionális és korai lárvális
életképtelenséget
okoz.
Az
elpusztult
kettős
mutánsok
súlyos
morfológiai
rendellenességeket mutattak. A két mechanizmus egyidejű inaktiválása negatívan hatott a
fonalféreg termékenységére is. Továbbá tapasztaltuk, hogy az apoptózis szintje autofág
mutánsokban megemelkedett a vad típuséhoz képest. Érdekes módon a gfp::lgg-1 autofág
marker konstrukció expresszója apoptózis mutánsokban nem tért el a vad típusétól.
Mindezek azt mutatják, hogy az autofágia és az apoptotózis redundánsan szükségesek a C.
elegans életképességéhez és egyedfejlődéséhez.
Az előzetes eredményeink alapján a továbbiakban olyan faktorokat kerestünk,
amelyek szabályozhatják az autofágiát és az apoptózist is. Az apoptózist moduláló bZip-szerű
CES-2/ATF-2 transzkripciós faktorok lehetséges kötőhelyét azonosítottuk a bec-1 gén
upstream szabályozó régiójában, és ez a szekvencia konzerváltnak bizonyult a különböző
Caenorhabditis fajok között. In vitro EMSA kísérletben a CES-2 és az ATF-2 is képes volt
specifikusan kötődni a bec-1 promoter vizsgált szakaszához. Továbbá in vivo rendszerben a
bZip∆_bec-1::gfp expressziója megnövekedett a vad típusú konstrukcióhoz képest. Mindezen
eredmények azt sugallják, hogy az ATF-2 valamint a CES-2 gátolják a bec-1 gén aktivitását, és
ez a szabályozás hozzájárulhat a sejtsors meghatározásához az egyedfejlődés során.
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15 Acknowledgements
First, I would like to thank my Supervisors Krisztina Takács-Vellai and Tibor Vellai.
They made it possible for me to prepare my work in their lab at the Department of Genetics,
showed me the wonderful face of science and especially genetics, taught me how to plan
and perform an experiment and led my thoughts with profitable consultations and progress
reports.
I thank prof. László Orosz, leader of the Classical and Molecular Genetics PhD
program, to support my doctoral work and teach me a lot from the marvelous side of
genetics.
I thank Éva Borsos, Tibor Kovács, Tímea Sigmond, Balázs Hargitai and Líz Pásztor, my
other co-workers from the Department of Genetics for the effective cooperation and that I
could learn a lot from them.
I would like to acknowledge Hilde Nilsen, Tanima Sengupta and Marlene Dengg
from The Biotechnology Centre in the University of Oslo, Norway for the fruitful
collaboration.
I also acknowledge all the members of the Vellai lab for the excellent working milieu
and humor. I have learned a lot about science and life during the years I have spent there.
I thank András Málnási-Csizmadia and Boglárka Várkuti, our collaborators from the
Department of Biochemistry, for the enjoyable and successful work.
I would like to say a big “thank you” to my Parents and my Siblings who always
support me when I need it. I thank all my friends who were with me in the hard days, and
last but not least thank to my girlfriend Dóra Olasz for your measureless support and love.
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