University of Groningen
Centrosomes and cellular stress responses
Hut, Henderika Magrietha Janet
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General Introduction
1
General Introduction
Stress responses
Mammalian cells exhibit complex cellular responses to genotoxic stress, such as cell cycle
checkpoints, DNA repair and apoptosis. Inactivation of these important biological events
may result in genomic instability and cell transformation (147). Genotoxic stress can be the
result of intrinsic processes (e.g. replication), and can be induced by radicals formed by
cellular metabolic processes. Genotoxic stress can also be induced by extrinsic sources
such as oxidative stress, irradiation or chemical agents. Another form of cellular stress, a
non-genotoxic form, is proteotoxic stress induced by heat shock. The best characterized
heat shock response is the induction of the heat shock proteins (HSP) which function as
molecular chaperones and that exert cell cycle regulatory and anti-cell death activities (126).
Thus, proteotoxic stress induced by heat shock in addition to genotoxic stress can affect
progression through the cell cycle (71).
Centrosomes, the microtubule-organizing centers (MTOC) of the cell, can be affected by
genotoxic stress (150; 159) as well as by proteotoxic stress (6; 12; 26; 96; 163; 164). The
aim of this thesis was to investigate in detail centrosome responses to different forms of
stress and to investigate what the consequences are for cells with affected centrosomes.
In the sections below an overview is given of cellular responses to genotoxic stress and to
proteotoxic stress. Subsequently a summary is given about centrosomes and the relation
between abnormal centrosomes and various kinds of stress. Finally the scope of this thesis
will be described. Unless otherwise mentioned, all the responses and characteristics we
describe here have been studied in mammalian cells.
Genotoxic stress
Checkpoints
Cells can respond to DNA damage by blocking cell cycle progression and therefore provide
the cell with sufficient time to repair the damage before DNA replication and cell division.
Such a block in cell cycle progression, called a cell cycle checkpoint, is a regulatory
mechanism that induces cell cycle arrest at characteristic time points in response to DNA
damage caused by internal or natural processes or by external agents (50; 63; 107; 130;
155; 165; 168; 169). Three major cell cycle checkpoints that respond to DNA damage are
the G1/S, the G2/M, and the intra-S-phase checkpoint. The last checkpoint temporarily
inhibits further DNA synthesis if the cells are in the S phase at the time of the DNAdamaging insult. Once the DNA damage is repaired, the regulating cellular machinery will
trigger the cell to continue the cell cycle (83; 130).
DNA damage checkpoints can be activated by various kinds of DNA damage, including UVlesions, replication errors, DNA cross-links, base damage and DNA strand breaks (50).
These checkpoints respond to different types of primary damage at different stages of the
cell cycle. Only lesions that cause the most serious damage, if not repaired before the next
stage of the cell cycle, will provide signals or the recognition of signals to activate the DNA
damage checkpoint. For example, DNA gaps or single strand breaks (SSB) will cause an
arrest at G1/S because failure to arrest would permit their conversion to double strand
breaks (DSB). In contrast, this checkpoint does not respond to unexcised dimers (yeast and
mammalian cells: (114; 151)) or a DSB (yeast: (140)), while the arrest at G2/M is
dramatically sensitive to even one DSB (yeast: (81)). Failure to arrest at G2/M while a DSB is
present would lead to a chromosome break and thus to the irreversible loss of a
chromosome fragment. Below, the three different checkpoints and a selection of players
involved in the checkpoints will be discussed.
11
Chapter 1
Arrest of the cell cycle at the G1/S checkpoint caused by DNA damage generally induces
increased cellular levels of the tumor suppressor p53. The presence of DNA strand breaks
leads to p53 induction, which requires the high-molecular weight phosphoinositide 3 (PI3)kinase ATM (Ataxia-Telangiectasia Mutated) (152). Through its transcriptional activity, p53
increases the expression of p21, a Cyclin-dependent kinase (Cdk) inhibitor that, in turn,
inhibits the Cdk2-CyclinE complex required for progression of the cell cycle from the G1 to S
phase. The intra-S-phase checkpoint also requires ATM activation, but is distinct from the
G1/S checkpoint. For example, the intra-S-phase checkpoint is not dependent on p53
activity, but it requires several other proteins in a complex such as BRCA1, Mre11, Nbs1
and Rad50 (167; 178). The G2/M checkpoint regulates the inhibition of the
dephosphorylation of Cdk1. Cdk1-CyclinB is required for the progression from G2 into M
phase. Key players in the G2/M checkpoint response are the kinases ATM and ATM-related
protein ATR (1), as well as checkpoint kinases Chk2 and Chk1 (2; 62). Cells that contain
diminished ATR (22; 172) or Chk1 (90) activity show a clear defect in the triggering of the
G2/M checkpoint in response to IR. However, cells deficient in ATM show no gross defects in
the triggering of G2/M arrest in response to IR (129; 174; 177). Likewise, cells deficient in
Chk2 are able to trigger the G2/M checkpoint in response to IR (53). Thus, conditions that
require the ATM/Chk2 pathway to elicit G2/M arrest have yet to be described. In Figure 1 an
overview is given of the major players involved in cell cycle checkpoints as mentioned in this
section.
Figure 1. Cell cycle checkpoints in mammalian cells.
An overview of the G1/S checkpoint, S-phase checkpoint, G2/M checkpoint and the spindle checkpoint in
mammalian cells. Solid arrows illustrate direct interactions and dotted arrows indicate that there are multiple
steps in between.
In addition to cell cycle checkpoints that respond to DNA damage, cells contain another
checkpoint: the spindle checkpoint. This spindle checkpoint does not respond to DNA
damage. The spindle checkpoint monitors the attachment of microtubules to kinetochores
and the tension at the kinetochores. In case chromosomes are not attached, or altered
tension is monitored, anaphase progression is inhibited and chromosomal instability due to
unequal segregation of chromosomes during cell division is prevented (82; 179). At the heart
of the spindle checkpoint is the kinetochore, a multi-layered proteinaceous complex that
assembles on the centromeric DNA of each chromosome. During mitosis, the kinetochore
mediates the interaction between the chromosome and spindle microtubules. At the very
beginning of prometaphase, immediately after the nuclear envelope breakdown (NEB),
kinetochores are not attached to microtubules. Subsequently, one kinetochore on a
12
General Introduction
chromosome captures microtubules from one spindle pole. When its sister kinetochore
captures microtubules from the other pole, the now bi-oriented chromosome moves to the
equatorial plane, the metaphase plate; this process is known as chromosome-congression
(145). Finally, all the chromosomes are attached through both kinetochores to microtubules
from two opposite spindle poles and aligned at the metaphase. The spindle checkpoint
ensures that, only when all the chromosomes are properly attached and aligned at the
equatorial plane, anaphase onset is triggered, allowing the splitting of sister chromatids and
their movement to each spindle pole (179). The spindle checkpoint regulates the APC
ubiquitin ligase (anaphase promoting complex, or cyclosome) that ubiquitinates M phase
cyclins (for cyclins: section ‘centrosomes and the cell cycle’) thereby regulating the exit from
M phase.
The spindle checkpoint is also defined as metaphase-anaphase checkpoint, mitotic
checkpoint, or as kinetochore checkpoint. However, although in contrast to what the name
‘spindle checkpoint’ would suggest, this checkpoint does not monitor defects in spindle
architecture itself like multipolar spindles (154). In our opinion, this checkpoint is better
called the kinetochore checkpoint. It first monitors the attachment of microtubules to
kinetochores and after all kinetochores are attached to microtubules it monitors the tension
at the kinetochores generated by the attached microtubules. Only when tension is generated
at all kinetochores, anaphase onset is triggered. However, the term spindle checkpoint is the
most widely used and therefore we will use spindle checkpoint in this thesis.
DNA repair
Checkpoint proteins arrest the cell cycle in the presence of DNA damage to allow the cell
time for repair. Moreover, the checkpoints are proposed to act not simply as a switch for
arrest, but also to directly recruit the DNA repair machinery or perhaps even play direct roles
in repairing DNA. For example, ATM, the main G1/S checkpoint regulator, is also involved in
regulating the repair of DNA damage by homologous recombination (HR) (117).
There are five main DNA damage repair pathways in mammalian cells (147; 158; 173).
These are the direct reversal (DR), mismatch repair (MMR), nucleotide excision repair
(NER), base excision repair (BER), and the recombinational repair pathways. DR is the
simplest repair mechanism in which one single enzyme removes the DNA damage in a one
step reaction. MMR, NER and BER rely on an intact complementary strand and can repair
various kinds of damage. Recombinational repair can be divided into HR and nonhomologous end joining (NHEJ) and is responsible for the repair of DSB and interstrand
DNA crosslinks. DSB, as well as SSB, can be caused by spontaneous hydrolysis of the
phophodiester bonds in the sugar backbone of DNA, by errors in completing DNA
replication, or by ionizing radiation (15).
In this thesis, only the recombinational repair pathways will be discussed in more detail, as
these repair pathways are most relevant to the experimental work performed. In HR, the
repair of one DSB in one chromatid uses the undamaged sister chromatid as a template. HR
almost exclusively takes place in the S and G2 phase of the cell cycle and is a multi step
process catalyzed by many gene products (HR reviewed in (121; 149)). Before genetic
exchange of the two homologous sequences, two DNA duplexes need to be partially
unwound in order to recognize each other by base pairing. These events are coordinated to
ensure that the event only occurs between large homologous DNA stretches. In this way, the
exact sequence of the genome is maintained (121; 149). However, during G1, homology can
solely be found in the other copy of the chromosome or in repetitive sequences. The
alignment is problematic and it is more important for the cell to immediately rejoin two
broken strands and maintain the configuration of the DNA than to spend time on finding the
exact homologous sequence. Therefore, in most cases DSB are repaired by NHEJ when
cells are in G1/G0. This pathway involves the direct rejoining of the broken, noncomplementary DNA ends of a DSB without the exchange of homologous sequences (NHEJ
reviewed in: (52; 55; 56; 131)). As a consequence, deletions and basepair substitutions will
arise, which makes this pathway error-prone. Thus, NHEJ does not faithfully restore the
original DNA sequence but does certify rejoining of DSB. Nevertheless, due to the high
13
Chapter 1
content of non-coding DNA in diploid somatic cells of multicellular organisms, these
mutations are expected to be tolerable in most cases (132).
Cell death expression / execution
When the above-mentioned DNA repair pathways fail, one possibility is that the cell
executes programmed cell death, referred to as apoptosis. This is a tightly regulated
phenomenon ensuring that cells that accumulate irreversible DNA damage do not replicate
and divide. Apoptosis is morphologically defined by cytoplasmic shrinkage, chromatin
condensation and nuclear fragmentation with chromatinolysis. Apoptosis is characterized by
biochemical events including the activation of caspases, DNA fragmentation, and the
permeabilization of the outer mitochondrial membrane (MMP), with the consequent release
of multiple death effectors, such as the caspase activator cytochrome C, from the
mitochondrial intermembrane space (9; 36; 143; 166).
Cells that do not initiate apoptosis may die by a necrotic form of cell death. Also, in case of
proliferating cells such as fibroblasts, DNA damaged cells may still remain biochemically
active, but they undergo permanent G1 arrest or terminal differentiation (24) that also
prohibits cells from generating progeny with a damaged genome.
It also has been demonstrated that after failure of the G2/M checkpoint, cells can undergo
mitotic catastrophe in M phase (16; 17). Whereas this was long considered as an
uncontrolled process, due to chromosomal damage, there are now (at least in Drosophila)
indications that this also may be a regulated process to prevent cytokinesis and hence
transfer of genetic errors to its progeny (159). Finally, cells that do undergo cytokinesis with
damaged DNA mostly will yield daughter cells with unbalanced numbers of chromosomes
and may undergo secondary apoptosis or necrosis. However, certain daughter cells may
stay alive and will carry mutations that eventually may lead to cancer (in somatic cells) or
that may cause inheritable diseases (in germ cells).
Proteotoxic stress
A variety of intrinsic and extrinsic stress conditions can be classified as proteotoxic stresses.
These can be defined as stresses that increase the fraction of proteins that are in an
unfolded state, thereby enhancing the probability of formation of potentially toxic intracellular
protein aggregates. A typical example of a proteotoxic, non-genotoxic stress is heat shock.
When cells are exposed to high temperatures, energy is absorbed throughout the cell,
damaging nearly all cellular structures and functions. Under normal physiological conditions,
an equilibrium of folded and unfolded proteins exists within the cell. Upon administration of
heat shock, this equilibrium is shifted, resulting in an increase in the fraction of unfolded,
denatured proteins that have lost part of their quaternary and tertiary structure. Hydrophobic
domains that are normally buried inside native proteins are now exposed. These
hydrophobic domains have a tendency to stick to other hydrophobic domains that will lead to
the formation of aggregates of denatured proteins within the cell and this formation of
aggregates is only partially reversible. Heat stress can lead to cell cycle arrest and cell
death, depending on the temperature and the duration of the exposure.
Heat causes a variety of morphological, biochemical, and molecular changes in the cell
(Table 1) (13; 60; 79; 85; 115; 127). Virtually all classes of macromolecules are affected by
heat shock. RNA and DNA at temperatures up to 48°C are reversibly damaged and this
minor damage does not to lead to a dysfunction of these macromolecules (58). Similarly,
although membrane lipids may be fluidized during hyperthermia, which depends on the
transition temperature of the lipids and the severity of the heat treatment, this process itself
is not the cause of heat-induced cell death (70). Numerous lines of evidence have led to the
conclusion that protein damage is the major molecular event leading to the biological effects
of heat stress (60). As elevated temperatures affect nearly all structures and organelles,
critical cellular targets are difficult to define and it is also not known which effect leads to the
induction of cell cycle arrest or cell death.
14
General Introduction
Cell cycle arrest
Cells accumulate in specific phases of the cell cycle after they have been exposed to heat
treatments. The major blocks are at the G1/S and the G2/M transitions. The accumulation at
specific phases depends on cell type and the phase in which the treatment was given. For
example, Chinese Hamster Ovary (CHO) cells heated in G1 exhibit a G1/S block, while HeLa
cells progress through the G1/S border after the same heat treatment, accumulate in S
phase and then undergo spontaneous premature chromosome condensation (PCC), leading
to cell death (94).
Table 1. Heat causes a variety of morphological, biochemical, and molecular changes
Organelle
Effects after heat stress
Membrane
Lipid fluidization
Aggregation integral membrane proteins
Collapse intermediate filament network
Disruption microtubuli
Appears fragmented
Appears fragmented
Protein denaturation
Polyribosomes disappear
Inactivation translation initiation factors
Inhibition protein synthesis
Preferential synthesis HSP
Swell
Decrease in number
Uncoupling respiration and oxidative phosphorylation
Protein denaturation
Decrease in number
Swells
Granular elements disappear
Electron-dense material appears
Relocation fibrous material
Inhibition rRNA synthesis and ribosome assembly
Inhibition DNA synthesis, RNA transcription, and RNA splicing
Protein denaturation and insolubilization
Specific transcription heat shock genes
Aggregation intermediate filaments
Accumulation denatured proteins
Cytoskeleton
Golgi apparatus
Endoplasmic reticulum
Protein translation machinery
Mitochondria
Lysosomes
Nucleolus
Nucleus
Centrosome and perinucleus
These data were based on references (13; 60; 79; 85; 128).
The absence or presence of the G1/S block may not be caused by the ability or inability to
activate the G1/S checkpoint. Rather, the response may simply be a passive process
because of protein damage that inhibits replication initiation. HeLa cells will still enter S
phase and initiate replication after heat shock, unlike CHO cells. If HeLa cells are further
heat stressed, S phase arrest will occur due to protein damage which inhibits replication
elongation and subsequently triggers PCC formation (93). Therefore, cell cycle progression
after heat shock may be purely the result of obstruction of essential processes, rather than
the elegant signaling found after DNA damage. Similarly, the accumulation of cells at the
15
Chapter 1
G2/M border (120) might be less dependent on signaling events, and merely caused by the
disturbance of processes needed to initiate mitosis, such as damage to centrosomes and
microtubules.
Thermotolerance
Thermotolerance is defined as a state of transient heat-resistance caused by exposure to an
initial heat shock followed by recovery at 37°C (so -called acute thermotolerance).
Thermotolerance can also occur during continuous mild heating, whereby initial cell death
gradually levels off and cells become resistant to further heating (so-called chronic
thermotolerance). Thermotolerance is strongly linked to the expression of HSP that
accumulate after the first heat shock or under chronic mild heating. Transcription of heat
shock genes is specifically induced under such conditions, while transcription of other genes
is inhibited by heat (108).
Although thermotolerance can develop in the absence of new HSP synthesis (60), several
observations have indicated that HSP play an important role. First, there is good agreement
between the kinetics of the transient heat-induced increase in HSP expression and the
kinetics of induction and decay of thermotolerance (21; 61; 86). Also, agents that induce
HSP, such as heavy metals and ethanol, induce thermotolerance (47; 171). Furthermore,
heat-resistant cell lines have elevated expression of one or more HSP (3; 80) and overexpression of some of the single members of the various HSP families has been shown to
result in a permanent thermoresistant state (75; 84; 116).
In mammalian cells, there are four major classes of HSP, namely the group of Hsp90,
Hsp70, Hsp60 and the group of small HSP such as Hsp27 (25). They all belong to the family
of ‘molecular chaperones’. Molecular chaperones are defined as proteins that bind to and
stabilize an otherwise unstable conformer of another protein. By controlled binding and
release, molecular chaperones facilitate the correct fate in vivo of the unstable protein. HSP
have the ability to bind specifically to exposed hydrophobic protein domains. Binding to
these denatured peptides prevents their sticking to other hydrophobic domains and as such
prevents excessive protein aggregation. The Hsp70 machine (i.e. Hsp70 plus a number of
regulators of its activity: (35)) has been thought to be especially crucial in the development
of thermotolerance (85).
Cell death
If checkpoints are not functional, cells may attempt to progress through the S phase, but
nuclear protein damage may obstruct DNA synthesis, leading to replication errors, lethal
chromatid aberrations and cell death (28). When cells enter mitosis with unresolved protein
damage (or when heated in mitosis), cells may undergo mitotic catastrophe or aberrant
divisions (112) that will mostly result in secondary apoptosis or necrosis of the daughter
cells. There are clear indications that accumulation of heat-unfolded proteins at centrosomes
are linked to these aberrant mitoses (112).
Centrosomes
The central question in this thesis is how do the centrosomes respond to various stresses
during the cell cycle. First, an overview is given of centrosome structure, duplication and
function. Then, the responses of centrosomes to various kinds of stress during the cell cycle
are discussed.
Centrosomes and the cell cycle
The duplication of centrosomes is linked in time to the DNA replication cycle and should
happen only once per cell cycle. Both processes involve cyclins, the regulatory proteins that
are present at high levels at specific phases in the cell cycle. Cyclins are needed for the
kinase activity of serine/ threonine kinases (Cdk’s) and each combination of Cdk-Cyclin is
specific for a certain cell cycle phase. For example, Cdk2-CyclinE regulates the progression
into the S phase as well as the initiation of the centrosome duplication (section ‘centrosome
duplication’).
16
General Introduction
Figure 2. A schematic structure of a centrosome.
The centrosome is composed of two centrioles, connected by interconnecting fibers and surrounded by the
PCM. Microtubules are nucleated from the PCM and the appendages of the mother centriole. Over 80 proteins
have been localized to this complex. Reproduced with permission from Nature Reviews Molecular Cell Biology
(30) copyright (2001) Macmillan Magazines Ltd. (www.nature.com/reviews).
Structure and biochemical composition
Centrosomes consist of two centrioles facing perpendicular to each other, surrounded by a
fibrous meshwork termed pericentriolar material (PCM). The two centrioles are barrel
shaped and each consist of nine triplet microtubules of ~400 nm in length (97). These stable
microtubules are composed of large numbers of polypeptides, including α-, β-, and δ-tubulin
(19), and centrin (5). The two centrioles differ both structurally (123) and biochemically (78)
and one is called the mother centriole and the other the daughter centriole. The mother
centriole is the oldest centriole and contains distal appendages. Some proteins, such as
cenexin (77) and Cep170 (44), only associate with the mother centriole, by localizing to the
distal appendages.
There are four classes of centrosomal proteins each of which are associated with a
particular function, including structural, microtubule-nucleating, anchoring, and regulating.
Examples of proteins from these four classes of centrosomal proteins are given in Table 2.
The structural proteins are required for maintenance of the morphology of centrosomes.
Microtubule-nucleating proteins assist the nucleation of microtubules with the minus ends to
the centrosome and are required for aster formation or microtubule growth from the
centrosome. Because of this function the centrosome is also referred to as the MTOC of the
cell. The microtubule-nucleating capacity is the classic function of centrosomes and this
capacity differs between the mother and daughter centriole. While both centrioles nucleate
microtubules, only the mother centriole anchors them, probably through its sub-distal
appendages containing ninein (133). Anchoring proteins are required to anchor microtubules
and regulating proteins to the centrosome and have at least two domains, namely an
enzyme-binding motif and a cellular localization motif. Anchoring proteins form the interface
17
Chapter 1
between microtubule-nucleating proteins and regulating proteins. Regulating proteins are
enzymatic proteins that regulate processes at the centrosomes. Regulating proteins include
kinases, phosphatases, signaling molecules and proteasomal components and can direct
the microtubule nucleating proteins (42). Cyclins and Cep135 are examples of regulating
proteins (118). In Figure 2 a schematic structure of a centrosome is shown and includes the
localization of some of the centrosomal proteins mentioned in Table 2.
Figure 3. The centrosome duplication cycle.
Centrioles are indicated as cylinders, and are numbered. Distal appendages are shown on centrioles 1 and 2,
indicating that these are mother centrioles. The line encircling the centrioles indicates the PCM. The dotted line
on the S phase centrosome B indicates that this centrosome is immature and that S phase centrosome A has
inherited the PCM. Reprinted from Cell, Vol. 105, Stearns, T., Centrosome duplication. a centriolar pas de deux,
417 - 420, Copyright (2001), with permission from Elsevier.
Centrosome duplication
The duplication of the centrosome is highly coordinated and can be divided into several
steps that will be discussed in the next sections. In short, duplication of centrosomes occurs
as follows. During G1 only one centrosome is present containing two centrioles: one mother
and one daughter centriole. At G1/S the two centrioles lose their orthogonal orientation and
each centriole forms a template for a newly synthesized procentriole. These procentrioles
elongate during S and so the centrosome contains four centrioles in this phase: one old
mother centriole, one old daughter centriole and two new daughter centrioles. In G2 phase
the link between the two old centrioles, also called parental centrioles, is broken. Each of the
two centrosomes matures, so that in the end both centrosomes will contain a mother
centriole, a daughter centriole, and PCM. At the onset of mitosis both centrosomes migrate
to opposite ends of the nucleus and each centrosome forms a mitotic pole of the bipolar
spindle. After completion of cytokinesis the two daughter cells each have inherited one
centrosome and the cycle starts over again. All phases of the duplication are controlled by
kinases and phosphatases as described below. A schematic overview is given in Figure 3.
Centriole duplication and elongation (G1/S phase)
It has been demonstrated in Xenopus that the initial round of centrosome duplication is
triggered by calcium/calmodulin-dependent protein kinase II (CaMKII), which is activated by
18
General Introduction
sudden increases in intracellular free calcium ions. This suggests that calcium oscillations in
the cell cycle are linked to centrosome duplication (99). In mammalian cells, the calciumbinding protein centrin is a member of this calmodulin super family (170).
Other factors have been identified that play a role in centrosome duplication and in Xenopus
oocytes and early embryos Cdk2-CyclinE activity is required for centrosome duplication.
Cdk2-CyclinE is also involved in centrosome duplication in mammals as outlined below. In
G1 phase, each cell has one centrosome and the association of phosphoprotein
nucleophosmin prevents its premature duplication. In early S phase, nucleophosmin is
phosphorylated by Cdk2-CyclinE and dissociates from the centrosome (74; 119). After
disappearance of nucleophosmin from the centrosome, the two centrioles lose their
orthogonal orientation by Skp1-Cullin-F-box (SCF) mediated proteolysis (Figure 3, G1/S
phase). The SCF ubiquitin ligase also ubiquitinates G1 cyclins and thereby regulates the
progression from the G1 into S phase. Each of the two centrioles forms a template for the
procentriole that is formed adjacent to the proximal wall and that elongates gradually during
the next phases (73) (Figure 3, S phase). Directly after nucleophosmin has disappeared
from the centrosome, Cdk2 activates mMps1p kinase to drive the centrosome duplication
process (37). The mMps1p kinase is localized to centrosomes during the entire cell cycle.
Cdk2 activity is not required for the centrosomal localization of mMps1p, but is required for
stabilization of mMps1p protein levels during S phase resulting in centrosomal accumulation
of mMps1p. This accumulation restricts the mMps1p kinase-dependent centrosome
duplication to the S phase (37).
Factors regulating parental centriole separation (G2 phase)
Just before G2 phase, the procentrioles elongate and the centrosome now consists of four
centrioles (Figure 3, S phase), namely two parental centrioles that are the old mother (Figure
3, centriole 1) and old daughter centriole (Figure 3, centriole 2) and two procentrioles (Figure
3, centrioles 3 and 4) forming the new daughter centrioles. The proximal ends of the two
parental centrioles are still connected by a proteinaceous link that consists of C-Nap1. A
constant delivery of microtubule subunits towards the centrosome-associated microtubule
minus ends determines the concentration of PCM components at the centrosome. In
addition, the microtubule network may play a role in the balance of kinases (NIMA family
member kinase Nek2) and phosphatases (PP1α, Cdc14A) that control regulatory processes
at the proteinaceous link (103). The dynamic interaction of Cdc14A with the centrosome may
help to generate this balance (95). Cdc14A interacts with interphase centrosomes and this
interaction is independent of microtubules or Cdc14A phosphatase activity. The amount of
Cdc14A is reduced at the centrosomes after entry into mitosis although it is not entirely
absent. Parental centriole separation is inhibited by the phosphatase PP1α, which is
believed to be a physiological antagonist of Nek2. At the onset of mitosis PP1α is inhibited
(138) and then Nek2 kinase is able to phosphorylate C-Nap1 (101). The regulation of CNap1 is cooperatively regulated by Nek2 and Aurora-A, a member of the Aik (Aurora/Ipl1related kinase) family (98). After phosphorylation of C-Nap1, the duplicated centrosome is
not connected by a proteinaceous link anymore but still the two, immature, centrosomes
localize close together.
Centrosome maturation (G2 phase)
Directly after breakage of the link between the two parental centrioles (Figure 3, centrioles 1
and 2), a difference is observed in protein content between the immature centrosomes. The
oldest centrosome (Figure 3, A), which contains the old mother centriole, has inherited the
PCM and is most mature. The newest centrosome (Figure 3, B), which contains the old
daughter centriole, has to acquire newly synthesized PCM as well as distal appendages at
the old daughter centriole to become the new mother centriole (Figure 3, G2 phase). εTubulin has been shown to localize only to the PCM of the oldest centrosome and later on to
the PCM of both centrosomes. At the same time ε-tubulin is acquired, cenexin is acquired at
the new mother centriole (19). In addition to the gain of new PCM, maturation of
centrosomes involves the phosphorylation of centrosomal proteins, and the increase of
microtubule-nucleating capacity (124).
19
Chapter 1
Migration to poles (M phase)
At the end of G2 phase, the two mature centrosomes localize just outside the nucleus. At this
moment the amount of γ-tubulin associated with the centrosome suddenly increases and this
γ-tubulin recruitment to centrosomes coincides with an increase of the centrosomal
microtubule-nucleation capacity during prometaphase (67). Withdrawal of the nuclear
membrane by the molecular motor dynein creates membrane tension, causing tearing and
resulting in NEB and subsequently in the release of several proteins such as nucleophosmin
and Ran-GTP. Nucleophosmin associates with the two centrosomes preventing them from
premature duplication. Ran-GTP, recruited and/or activated by Polo-like kinase Plk1 (54),
causes increased microtubule nucleation from the centrosomes. Each centrosome moves to
one side of the nucleus. The formation of the spindle by interaction of microtubules and
chromosomes and the trigger to anaphase onset is described in the section ‘checkpoints’. At
the end of telophase, the two daughter cells are still connected by the midbody, a
cytoplasmic bridge consisting of microtubules.
Cytokinesis completion
Each of the two daughter cells inherits one centrosome and while the mother and daughter
centriole of one centrosome are always close together during the rest of the cell cycle, they
separate into individual units just after telophase. The mother centriole remains almost
stationary near the geometrical center of the forming daughter cell and bears the
microtubule aster, while the daughter centriole is more mobile and only nucleates a few, if
any, microtubules (133). The mother centriole then suddenly moves towards the midbody
(133). The cytoplasmic bridge narrows, the microtubules within the bridge depolymerize, and
cytokinesis is completed when the mother centriole moves back to the daughter centriole
(134) after which the movements of the daughter centriole reduce (133). Remarkably, the
movement of the monocentriolar unit described here is required for the fidelity of the
completion of cytokinesis.
Centrosome function in mitosis
Although centrosomes play an important role in the M phase, the centrioles of the
centrosome are not essential for assembling spindles (49), segregating chromosomes (49),
directing cell division, or forming centrosomes. In the absence of centrioles, these processes
depend on self-organization of microtubules (48). In the absence of centrioles, centrosomal
components, organelles, and microtubules interact with each other to produce polarized
microtubule arrays that resemble centrosomes. In addition, chromosomes and microtubules
can influence each other’s behavior to form spindles. However, the absence of centrioles
often results in cytokinesis failure (49) because chromosomes persist in the midbody (66)
and because cytokinesis does not complete if the mother centrioles do not move towards the
midbody (134; 146).
Although the centriole of the centrosome is dispensable for mitosis, its presence increases
the fidelity of mitosis (144; 146). The centriole induces the formation of centrosomes at
specific times and in specific places thereby increasing the accuracy and speed of the
processes the centrosome directs.
Centrosome function in interphase
As described in the section ‘centrosomes and the cell cycle’, the classic function of
centrosomes is their role as the MTOC in the cell. Through its nucleating and anchoring
capabilities, the centrosome organizes the cytoplasmic microtubule complex. However, a
typical interphase microtubule array can be formed in the absence of a centrosome. Under
these conditions, microtubules are nucleated randomly within the cytoplasm and organized
into an aster by the action of multivalent microtubule molecular motors such as dynein.
These a-centrosomal microtubule arrays support all normal intracellular trafficking, but it is
not known whether they support all microtubule-mediated functions (144).
While the centrosome is dispensable to the cytoskeleton, this organelle is essential for the
progression from G1 into S phase (31; 49). It is not known whether this is because the
20
General Introduction
centrosome is part of a checkpoint pathway (110), or because the centrosome is required
simply to catalyze reactions needed for progression into the S phase (144).
Centrosomes and genotoxic stress
Under normal conditions, centrosome duplication and DNA replication are coupled.
Centrosomes are essential for progression into S phase, the DNA synthesis phase, and play
a role in increasing the fidelity of cytokinesis. Remarkably, in the presence of damaged DNA
during mitosis, centrosomes appear to be important target organelles for regulating cell
division. For example in Drosophila embryos, the presence of damaged or incompletely
replicated DNA during mitosis triggers the dissociation of several components of the γtubulin ring complex (γ-TuRC) from the centrosome, leading to loss of microtubule
nucleation and to defects in spindle assembly. Anastral spindles are formed that are unable
to segregate the DNA. These features occur in wild-type embryos treated with replication
inhibitors or DNA-damaging agents (150; 159). Various types of agents that can cause DNA
lesions trigger the mitosis-specific centrosome inactivation, e.g. DNA replication inhibitors
aphidicolin and hydroxyurea, DNA-damaging agents such as UV radiation and X-rays, the
radiomimetic drug bleomycin, Topoisomerase I or II inhibitors camptothecin, etoposide VM26 and ICRF-193, and also injection of phage DNA that was restriction-digested, singlestranded or supercoiled (150; 159).
A null mutation in the Drosophila Chk2 tumor suppressor homologue (DmChk2/Dmnk)
blocks the mitosis-specific centrosome inactivation response to DNA lesions and also
prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to
increased DmChk2/Dmnk localization to the centrosome and spindle microtubules.
DmChk2/Dmnk is therefore essential for a ‘mitotic catastrophe’ signal that disrupts
centrosome function in response to genotoxic stress and ensures that aneuploid nuclei are
eliminated from the embryonic precursor pool (159).
Whether a mitosis-specific centrosomal inactivation pathway also exists in mammalian cells
was not known at the start of this project and one part of this thesis is dedicated to
investigate this.
Centrosomes and proteotoxic stress
Proteotoxic stress also leads to centrosomal abnormalities, although this might be an
indirect response. Misfolded proteins that arise after heat shock are able to aggregate with
each other or with other proteins in the cell. Misfolded proteins were shown to accumulate at
the centrosomes in interphase cells (164). These so-called aggresomes (57) may enable
cells to coordinately regulate and dispose of protein damage at specific cellular sites and
hereby preventing random protein aggregation and cellular dysfunction. However, it is not
unlikely, although not proven yet, that protein aggregates at the centrosome may also lead
to dysfunction of the centrosomes in cell cycle progression.
In Drosophila embryos, heat shock leads to severe defects in centriole organization
specifically when cells are heated in mitosis (26). Cellular consequences of these heat shock
affected mitotic centrosomes have not been studied in this system.
At the start of this project, centrosomal effects of heat shock specifically given during mitosis
had not been studied in mammalian cells. However, during interphase, centrosomal
abnormalities caused by heat treatments were observed. These centrosomal abnormalities
include increases in the electron-dense material comprising the PCM, disappearance of
centrosomal proteins such as γ-tubulin and pericentrin, and disruption of centriole walls (6;
12; 96; 163; 164). In addition, abnormalities in the first mitoses post-heating were reported
including the appearance of multipolar mitotic spindles (112; 162).
Protection of centrosomes from protein damage during heat treatments has been
demonstrated for centrosomes in interphase because thermotolerant interphase cells display
a more rapid recovery from heat-induced damage to the centrosome structure and
centrosome function (12; 163). However, a protective role of HSP to protein damage at the
21
Chapter 1
centrosomes of mitotic cells was not investigated and remained elusive at the start of this
thesis.
Nevertheless, in untreated conditions a role for HSP in centrosome function has been
suggested. For example, Hsp90 is localized at the centrosome throughout the cell cycle, at
different stages of development in Drosophila and vertebrates. Disruption of Hsp90 function
by mutations in the Drosophila hsp90 gene or treatment of mammalian cells with an Hsp90
inhibitor, results in abnormal centrosome separation and maturation, aberrant spindles and
impaired chromosome segregation (76). Another example is that Hsp73, the constitutive
form of Hsp70, is intimately associated with the centrosome during all stages of the cell
cycle in untreated conditions, suggesting that Hsp73 is involved in centrosome function (11;
142). It was speculated that Hsp73 participates in the process of centrosome assembly.
Following the duplication of centrioles during S phase, Hsp73 mediates the assembly of the
proteins that comprise the PCM. This is consisting with the finding that Hsp73 has a role in
the return of centrosomal structure and microtubule-nucleating function following heat shock.
Furthermore, these authors suggested that the presence of Hsp73 at the centrosome during
other stages of the cell cycle might facilitate the movement of proteins into and out of the
organelle (12).
Centrosomes and cancer
One characteristic phenotype of many cancer cell lines is the presence of multiple
centrosomes (10; 23; 87; 139; 148). Centrosome amplification and abnormalities occur
exclusively in aneuploid, but not diploid cancer cell lines, and correlates with numerical
chromosomal alterations (43). In addition to abnormal centrosome numbers, centrosomes in
human tumors display also significant alterations compared to centrosomes in normal tissue.
These alterations include supernumerary centrioles, excess PCM, disrupted centriole barrel
structure, unincorporated microtubule complexes, centrioles of unusual length, accumulation
of inappropriate phosphorylation of centrosomal proteins, and increased numbers of MTOC
nucleating unusually large arrays of microtubules (88; 89; 135; 136). However, it remains
unclear whether multiple centrosomes are merely a consequence of the genomic instability
in transformed cells, or whether they also play a driving force in the process of
carcinogenesis.
In particular it has been suggested that important tumor suppressors like p53 or BRCA1 are
associated with centrosomes. It can therefore be speculated that their loss of function may
amongst others result in a defective regulation of centrosome duplication, either in the
absence or in the presence of (induced) DNA damage. However, both proteins are also
otherwise linked to genomic instability, namely via checkpoint control or DNA repair. Hence
it needs to be tested whether the abnormal number of centrosomes in cells lacking BRCA1
or p53 is due to a direct loss of control on centrosomes or rather due to an indirect result of
the involvement of these tumor suppressors in cell cycle regulation and DNA repair.
22
Table 2. Centrosomal proteins, categorized by function.
Localization in centrosome
Remarks
Ref
Structure
23
α-tubulin
Centriole
αβ-dimers stack head-to-tail, aggregate laterally and form cylinder.
(51)
β-tubulin
Centriole
αβ-dimers stack head-to-tail, aggregate laterally and form cylinder.
(51)
Cenexin
Mother centriole
Marker for maturation: it is acquired by immature centriole at G2/M transition.
(77)
Centrin
Distal lumen of centrioles
Probably involved in centrosome reproduction cycle. Calcium-binding protein.
(125)
C-Nap1
Proximal (closed) centriole end;
proteinaceous link between
parental centrioles
Key component of dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.
(C-Nap1 = Cep250)
(41; 102)
δ-tubulin
Between centrioles and between
centrosomes
Dyn2
Centrioles and PCM
Directly interacts with γ-tubulin. Dyn2 (Dynamin 2) is dynamic exchangeable component of centrosome
and is participant in centrosome cohesion. Dyn2 is a GTPase.
(160)
ε-tubulin
PCM
Marker for maturation: it associates with oldest centrosome.
(19)
Odf2
G0/G1 mother centriole, G1/S
mother and daughter centriole
May be involved in maturation of daughter centrioles. (Outer dense fiber 2)
(111)
(19)
General Introduction
Remarks
Ref
Anchoring (subdistal appendages of mother centriole are major microtubule-anchoring structures of centrosome)
PCM
Anchors CK1δ and probably also CK1ε. The association between centrioles and AKAP450 is critical for
centrosome integrity and reproduction. (A-Kinase Anchoring Protein; = AKAP350 = CG-NAP)
(65; 153)
Cep110
Mother centriole
Marker for maturation: it appears at daughter centrosome during telophase-G1 transition.
(122)
EB1
Mother centriole
Anchors cytoplasmic MT minus ends to subdistal appendages of mother centriole. (End-binding protein 1)
(92)
Kendrin
Centrosome
Anchors γ-tubulin. Kendrin binds DISC1 (Disrupted-In-Schizophrenia 1). N-terminal regions of Kendrin share (38; 105)
significant sequence homology with pericentrin.
Ninein
Mother centriole and PCM
Marker for maturation: it appears at open end of daughter centrosome during telophase-G1 transition;
Proposed to have dual role as MT minus-end capping and anchoring protein; Promotes MT nucleation by
docking γ-TuRC at centrosome; Can be phosphorylated by Aik and PKA.
(8; 20; 27;
106; 122)
Nlp
Mother centriole
Is coordinately regulated at G2/M by Plk1 and Nek2 and is displaced form centrosome upon mitotic entry. It
binds γ-tubulin.
(141)
Pericentrin
PCM
Anchors γ-TuRC at spindle poles, thereby playing a role in MT nucleation.
(180)
γ-tubulin
PCM
Recruits αβ dimers.
(51)
Ran
Centrosome
Centrosomal interaction mediated by AKAP450. When AKAP450 is delocalized from centrosome,
Ran is also delocalized, and MT regrowth or anchoring is altered. Ran is a GTPase.
(64)
RanBP1
Centrosome
RanBP1 (Ran-binding protein 1) is major effector of Ran. Overexpression of RanBP1 induces centrosome
splitting during mitosis, which requires microtubule integrity and Eg5 activity.
(29; 113)
TCP-1
Centrosome
Microtubule regrowth.
(11)
24
AKAP450
MT nucleation
Chapter 1
Localization in centrosome
Localization in centrosome
Remarks
Ref
Aik
Spindle pole (prophase to
anaphase)
Possibly involved in centrosome function such as chromosome segregation or spindle formation.
Aik3
Mitotic centrosome (anaphase Suggested to play role in centrosome function at later stages of mitosis.
to cytokinesis)
APC10/ Doc1
Mitotic centrosomes
APC10/Doc1 (APC subunit 10) may be one of core subunits of APC, the ubiquitin ligase which
specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclinB.
(72)
Aurora-A
Centrosomes (from S phase
after centriole duplication till
early G1)
Involved in centrosome separation, duplication, maturation, bipolar spindle assembly and stability.
Aurora-A is potential oncogene. (Aurora-A = Stk15); Phosphorylates Cdc25B at centrosome during
mitosis and might locally participate in control of mitotic onset. NB: Cdc25B phosphatase is activator of
Cdk’s at mitosis (e.g. Cdk1-cyclin B1).
(33; 34)
hCdc14A
Centrosomes
hCdc14A is phosphatase with dual specificity: it dephosphorylates hCdh1 and activates APC(Cdh1),
but it does not affect APC(Cdc20) activity; NB: APC(Cdh1) ubiquitinates mitotic cyclins, allowing mitotic
exit.
(7)
Cdc20
Centrosomes
Cdc20 is activator of APC/C and has rapid turnover at spindle poles.
(59)
Cdk1
Centrosomes (elevated levels Modification of centrosome bringing about formation of mitotic spindle. (Cdk1 = p34cdc2)
G2/M)
(4)
Cep135
PCM
Role in centrosomal function of organizing microtubules.
(118)
Cep170
Interphase mother centriole
Marker for maturation: localizes to subdistal appendages, typical of the mature mother centriole. Thus,
one centriole during G1, S, and early G2, but two centrioles during late G2 phase.
(45)
CK1δ
Interphase centrosome
Contribution to cell cycle progression (vesicular trafficking, spindle formation). (Casein kinase 1)
(104; 153)
CK1ε
Interphase centrosome
Contribution to cell cycle progression (vesicular trafficking, spindle formation).
(104; 153)
Regulating
(68)
(69)
25
General Introduction
Remarks
Ref
Cul1
Centrosomes
Member of SCF complex.
(39)
CyclinE
Centrosomes
Has modular centrosomal-targeting domain essential for promoting S phase entry in Cdk2-independent
manner.
(100)
Erk
Centrosomes
Links extracellular signaling to centrosome dynamics by Nek2A. Interacts with Nek2A via conserved Erk2 (91)
docking site.
Katanin
PCM
Regulates number of microtubule ends in spindle (it is ‘MT-severing’). Katanin's activities are segregated (14; 46)
into subunit (p60) that possesses enzymatic activity and subunit (p80) that targets the enzyme to
centrosome.
NM23
Centrosomes
NM23-H1 is putative tumor suppressor. It associates with Aurora-A/STK15.
(32)
Nek2
Centrosomes
Centrosome separation at the G2/M transition. (NIMA (never in mitosis A)-related kinase)
(40)
Nucleophosmin
Mitotic centrosome (till
anaphase)
Involved in nucleolar assembly, centrosome duplication, ribosome assembly, and transport. Centrosomal (18; 176)
localization and function regulated by Nek2A. (=NPM/B23 = NO38 = Numatrin)
p53
Mitotic centrosome
ATM is essential for p53 centrosomal localization (required for activation of post mitotic checkpoint after
spindle disruption, thus for inhibiting DNA reduplication). P53 is tumor suppressor.
Plk1
Centrosome
Regulator of chromosome segregation, mitotic entry, and mitotic exit; Plk1 (= Polo; = Plx1) may play
(54)
centrosomal roles through recruitment and/or activation of Ran/RanBPM (Ran-binding protein in MTOC).
PML
Centrosomes
Direct role in control of centrosome duplication through suppression of Aurora-A activation to prevent
centrosome reduplication. (promyelocytic leukemia gene)
(175)
RASSF1A
Mitotic centrosomes
Interacts with Cdc20 resulting in inhibition of APC activity and thereby regulates mitotic progression.
RABP1 (RASSF1A-binding protein 1 = RAS association domain family protein 1A = C19ORF5) is
required for recruitment of RASSF1A to spindle poles and for inhibition of APC-Cdc20 activity during
mitosis. RASSF1A is tumor suppressor.
(156; 157)
Skp1
PCM
Role in proper chromosomal segregation. Member of SCF complex.
(39; 137)
(109; 161)
Chapter 1
26
Localization in centrosome
General Introduction
Aim of the thesis
A well-established role for centrosomes is to increase the fidelity of mitosis, although
centrosomes themselves are not required for progression through mitosis. Only recently it
has been demonstrated that centrosomes also play a novel role in responses of cells to
various kinds of stress. Drosophila embryos respond to genotoxic stress by a mitosisspecific centrosomal inactivation pathway. This pathway is dependent on DmChk2. The aim
of this thesis was to investigate whether there exists a comparable centrosomal inactivation
pathway in mammalian cells. Therefore we investigated in detail whether and how various
stress events affect mitotic centrosomes in mammalian cells and what the consequences
are for dividing cells with altered centrosomes.
27
Chapter 1
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36
General Introduction
37
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38