Identifying Factors Involved in Chromosome
Movement During Prophase I of Meiosis in
Caenorhabditis elegans
Jasmin Chan Hanafi
Department of Biology
McGill University
Montreal, Quebec, Canada
22 July, 2013
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science
© Jasmin Chan Hanafi, 2013
Table of Contents
Table of Contents ..................................................................................................... i
List of Figures ......................................................................................................... ii
List of Tables ......................................................................................................... iii
Abstract .................................................................................................................. iv
Resumé .................................................................................................................... v
Acknowledgements ............................................................................................... vii
Chapter I: Introduction and Literature Review ....................................................... 1
I.1 – Overview of Meiosis .................................................................................. 1
I.2 – Homologous Chromosome Recognition, Pairing and Recombination
During Prophase I ............................................................................................... 2
I.3 – C. elegans as a Model System to Study Meiosis ........................................ 3
I.4 – Meiosis Specifics in C. elegans .................................................................. 4
I.5 – Cytoskeleton ............................................................................................... 6
I.6 – Regulation of Cytoskeleton ........................................................................ 7
I.7 – Molecular Motors ....................................................................................... 9
I.8 – Regulation of Motor Proteins ................................................................... 10
I.9 – Interaction Between Different Cytoskeleton Networks ............................ 11
I.10 – The Nucleoskeleton ................................................................................ 12
I.11 – Research Proposal ................................................................................... 13
Chapter II: Materials and Methods ....................................................................... 14
II.1 – Plates and Culture .................................................................................... 14
II.2 – C. elegans culture .................................................................................... 14
II.3 – Feeding RNAi and Scoring ..................................................................... 15
II.4 – Time-lapse Microscopy ........................................................................... 15
II.5 – Immunostaining ....................................................................................... 16
Chapter III: Results ............................................................................................... 17
III.1 – Selecting RNAi Candidates ................................................................... 17
III.2 – Controls .................................................................................................. 18
III.3 – Results of RNAi Screen ......................................................................... 18
Chapter IV: Discussion and Future Directions ..................................................... 20
IV.1 – Candidate Gene syp-2 Validates RNAi Screen Methodology ............... 20
IV.2 – emb Phenotype Not Necessarily Linked to Lack of Chromosome
Movement Phenotype ....................................................................................... 20
IV.3 – Future Directions ................................................................................... 21
Chapter V: Conclusion .......................................................................................... 24
Figures ................................................................................................................... 25
Tables .................................................................................................................... 34
References ............................................................................................................. 50
i
List of Figures
Figure 1 Phases of meiosis .................................................................................... 25
Figure 2 Diagram illustrating gonad structure ...................................................... 26
Figure 3 Chromosome end attachment to nuclear envelope ................................. 27
Figure 4 Progression of chromosome movement and pairing .............................. 28
Figure 5 Proposed model for role of microtubules in regulating nucleus
positioning............................................................................................................. 29
Figure 6 chk-2 RNAi causes loss of PLK-2 foci and patches ............................... 30
Figure 7 Comparison of WT vs no movement phenotypes .................................. 31
Figure 8 syp-2 RNAi results in clear defects in the gonad.................................... 32
Figure 9 Break down of positive candidates ......................................................... 33
ii
List of Tables
Table 1 List of all candidate genes........................................................................ 34
Table 2 List of positive candidates ....................................................................... 40
Table 3 List of positive hits with emb phenotype ................................................. 45
Table 4 List of positive hits with him phenotype .................................................. 45
Table 5 List of negative hits with emb phenotype ................................................ 46
Table 6 List of positive hits with roles in MT motor activity ............................... 47
Table 7 List of genes untested due to unavailability............................................. 48
iii
Abstract
Meiosis is a reductional cell division that produces haploid gametes and uniquely
allows the introduction of genetic diversity via crossover recombination between
homologous chromosomes. Any defect during this process could lead to the
non-disjunction of chromosomes, which in turn leads to aneuploidy in the
resulting progeny, a condition that is generally lethal but in certain cases results in
serious developmental abnormality. In C. elegans, at the onset of meiosis,
chromosomes condense and cis-acting regions near each chromosome end called
pairing centers recruit zinc-finger proteins which help chromosomes associate
with nuclear envelope bridge proteins. These bridge proteins are in turn linked to
the cytoskeleton network. This association is important to facilitate chromosome
clustering and homology search. Once chromosomes are properly homologously
paired, the process of division continues until eventually four haploid cells are
produced. Though the successful coordination and regulation of each step in
meiosis is critical for the survival of a species, many components of the process
remain unclear.
During prophase I, chromosome movement resulting in proper
homologous pairing is controlled and regulated in a manner that is not well
understood. The objective of my research therefore, is to try and identify factors
involved in this chromosome movement. To do this, a candidate RNAi screen of
482 genes was conducted and 156 genes were positively identified as having a
lack of chromosome movement. Since any mistakes in pairing and subsequent
stabilization of homologous chromosomes could lead to non-disjunction and
possible embryonic lethality (emb) from loss of an autosome or a high incidence
of males (him) from loss of the X chromosome, positive candidates were also
screened for emb and him. Of the 156 positive candidates, 24 were also positive
for emb and 1 was additionally positive for him. These candidates present many
possibilities for further validation and characterization in future projects.
iv
Resumé
La méiose est une division cellulaire réductionnelle qui produit des gamètes
haploïdes et permet d'une façon unique l'introduction de diversité génétique à
travers la recombinaison entre les chromosomes homologues. Tout problème dans
le processus peut causer une impossibilité de séparation entre les chromosomes,
ce qui à son tour cause l’aneuploïdie dans la génération suivante, une condition
qui est généralement mortelle, mais résulte en anomalie dans le développement
dans certains cas. Les chromosomes de C. elegans, au tout début de la méiose, se
condensent et les régions “cis” à la fin de chaque chromosome appelées centres
paires recrutent les protéines avec des doigts de zinc qui aident les chromosomes
associer avec le pont de protéines sur l'enveloppe nucléaire. Le pont est connecté
au réseau cytosquelette. Cette association est importante pour la facilitation du
regroupement des chromosomes et la recherche de chromosomes homologues. À
la retrouvaille des chromosomes homologues, le processus de division continue
jusqu'à ce que quatre cellules haploïdes soient produites. Même si le succès de la
coordination de chaque étape de la méiose est critique pour la survie des espèces,
certain détails du processus restent inconnus.
Durant la prophase I, le mouvement des chromosomes qui résulte dans le
propre couplement des chromosomes homologues est contrôlé et régulé d'une
manière encore inconnue. L'objectif de mes recherches est donc d'identifier des
facteurs associés dans ledit mouvement des chromosomes. Pour accomplir ce but
un écran de ARNi avec 482 gènes comme candidates a été mené et 156 gènes ont
été positivement identifiés pour une manque de mouvement des chromosomes.
Comme tout problème de formation des couples de chromosomes ainsi que dans
la stabilisation des chromosomes homologues qui suit peut causer de la nondisjonction et possible mort embryonnaire (emb) suite à la perte d'un autosome ou
une haute incidence de males (him) causé par la perte du chromosome X, les
candidats out aussi été examinés pour emb et him. Des 156 candidats positifs, 24
ont aussi été positifs pour emb et un candidat a été additionnellement positif pour
v
him. Ces candidats se présentent comme une source de futures recherches de
validation ainsi que de caractérisation.
vi
Acknowledgements
This thesis is the product of many years worth of blood, sweat, tears, and love,
and would not have been made possible without the invaluable help and guidance
I received along the way.
I would first like to thank my supervisor Dr. Monique Zetka for first
inspiring me to delve into the realm of meiosis research and for giving me the
opportunity to work in her lab. I owe thanks, too, to my supervisory committee
members Dr. Gary Brouhard and Dr. Joseph Dent for their advice in helping to
refine and better streamline my project.
I would also like to thank the past and present members of the Zetka lab
for all their instrumental support and advice throughout my years in the lab. I
would additionally like to thank Sara Labella and Ka-lun Law for their neverending patience in teaching me techniques, especially when I first started out,
Florence Couteau for her help brainstorming and to Buget Saribek for her goodhumoured company on my otherwise lonely weekend vigils. I am grateful, too, to
have had the pleasure of working with the undergraduates, especially Anja
Boskovic, Camille Delouche and Kevin Dick. Huge thanks to Aleksandar Vujin
for his insights, editing, above-and-beyond support, and friendship during our
time as Master’s students together.
I would also like to thank Dr. Richard Roy and the members of the Roy
lab for their help and wisdom. In particular, thank you Yu Lu for all the much
needed advice for my thesis.
A big thank you to my “bio-sister” Milena Popova for her continued help
and advice with all things biology and thesis related, her help translating, and her
unwavering encouragement and friendship. Thanks to Adam Mitchell for help
with proof-reading and photo/figure editing.
Finally, I would like to express my love and thanks to my family for fully
supporting my decision to pursue a Master’s degree despite the distance. I also
owe special thanks to Bryan Too for being my rock and giving his love and
support.
vii
Chapter I: Introduction and Literature Review
Meiosis is a specialized cell division process that differs from mitosis in that it
reduces a diploid cell to haploid gametes through two successive divisions after
only a single round of DNA replication. It also uniquely allows for the shuffling
of genes into novel configurations through crossover recombination between
homologous chromosomes. This is an important mechanism for ensuring bivalent
biorientation during metaphase I, which is necessary for proper segregation, and
also for the generation of genetic diversity. For proper crossover recombination to
take place however, chromosomes must first be homologously paired. In
C. elegans, pairing centers on each chromosome end recruit adaptor proteins
which help associate chromosomes with the nuclear envelope and by extension,
the cytoskeleton network. Chromosomes then begin to cluster at one end of the
nucleus in a bouquet formation which further facilitates chromosome homology
search and alignment. Any defect during this process could lead to the
non-disjunction of chromosomes, which in turn leads to aneuploidy in the
resulting progeny, a condition that is generally lethal but in certain cases results in
serious developmental abnormality (such as Down syndrome/trisomy 21 or
Edwards syndrome/trisomy 18 in humans). Thus, successful coordination and
regulation of each step in meiosis is critical for the survival of a species. Despite
its essential role in sexually reproducing organisms, many components of the
process remain unclear; one critical question is how, during prophase I,
chromosome movement resulting in proper homologous pairing is controlled and
regulated. This question was the focus of my research and I conducted a screen to
try and identify factors involved in chromosome movement.
I.1 – Overview of Meiosis
Like mitosis, meiosis in diploid cells is preceded by one round of DNA
replication. This process gives rise to identical sister chromatids linked together
by the cohesin complexes. The cell then enters the first phase of division proper,
called prophase I. During this phase, homologous chromosomes find and align
1
with one another in a process called pairing resulting in parallel juxtaposition of
their chromosome arms. This is followed by a more intimate association called
synapsis, which is mediated by a proteinaceous structure called the synaptomenal
complex (SC), and after which recombination can take place between homologous
chromosomes, introducing genetic variation and forming physical bridges called
chiasmata which link homologous chromosomes and help keep them together
through metaphase I, when the SC itself has depolymerized. At metaphase I,
kinetochore microtubules from centrioles at either end of the cell attach to their
respective kinetochores and align the homologous chromosomes along the
division plane. During anaphase I, the kinetochore microtubules begin to shorten,
pulling the homologous pairs apart to each spindle pole. In telophase I the cell
completes division: the microtubule spindles disappear, the nuclear membrane is
reformed around each set of chromosomes which begin to uncoil, and cytokinesis
occurs, forming two distinct daughter cells. Without further DNA duplication, a
second, equational division similar to mitosis, called meiosis II, occurs. Like
meiosis I, meiosis II has 4 main stages: prophase II, metaphase II, anaphase II and
telophase II. It is during this second division that sister chromatids separate and
segregate, producing haploid gametes.
I.2 – Homologous Chromosome Recognition, Pairing and
Recombination During Prophase I
In order for proper chromosome segregation to take place during the first
meiotic division, chromosomes must first find and recognise their homologous
partner, align along their lengths, and stably pair via physical linkages. Prophase I
is where the key events of homologue recognition, pairing and recombination
occur, and it is further divided into 5 stages (Figure 1). Prophase begins with the
leptotene stage, when chromosomes, having already been duplicated, begin to
condense, axial elements begin to develop, chromosome ends move to the nuclear
envelope (NE) and associate with NE transmembrane proteins and chromosomes
begin to cluster at one end of the nucleus in a bouquet formation1. Next is the
zygotene stage, when homologous chromosomes find, recognise and pair with one
2
another and the SC forms along the length of paired homologs to structurally
reinforce pairing (a process known as synapsis)1, The pachytene stage then
follows, when further stabilization of pairing occurs either through crossover
recombination, which results in the formation of physical linkages known as
chiasmata between homologues, or by pairing at cis-acting loci on the
chromosomes called pairing centers (PC)1. After this comes the diplotene stage,
when the SC is degraded leaving homologs attached by just the chiasmata1.
Finally there is the diakinesis stage, when the 4 chromatids (tetrads) are clearly
visible, the nuclear membrane disintegrates and the meiotic spindles begin to
form1.
I.3 – C. elegans as a Model System to Study Meiosis
In order to delve deeper into the underlying mechanisms that control
chromosome movement during homology search in prophase I, a suitable model
system must be selected. C. elegans is one such system as it has a short life cycle
and is transparent, which allows for adult worms to be quickly grown for live
imaging of gonad nuclei undergoing meiosis. The gonads are a syncytium of
nuclei arranged in a spatio-temporal manner such that at the distal end of the
gonad there are mitotic cells, and these progress on to become meiotic cells at the
leptotene/zygotene stage in a region of the gonad known as the transition zone
(TZ). Meiotic cells continue to extend proximally with a gradient of meiotic
progression through prophase I until finally, at the proximal end, gametogenesis
occurs (Figure 2). The benefit of this arrangement is that meiotic progression can
be observed in a population of synchronised cells (i.e. all the nuclei undergoing
the same phase in meiosis will already be physically grouped together) and that
nuclei at the leptotene/zygotene stage can easily be identified as having a
crescent-shape due to chromosome clustering. It has also been previously shown
that a fluorescently tagged version of polo-like kinase PLK-2 is a robust meiotic
marker of all PC ends at the NE as well as an indicator of proper chromosome
movement and pairing, further making collection of data feasible2.
3
Furthermore, the process of RNA interference (RNAi) can be initiated by
introducing double-stranded RNA (dsRNA) into the worm; the dsRNA targets
and specifically degrades the endogenous messenger RNA (mRNA) which shares
its sequence, effectively inactivating the gene of interest with few off-target
effects3-5. The required dsRNA can be introduced into the worm via a variety of
methods: injection of dsRNA or plasmid DNA expressing dsRNA under the
control of a C. elegans promoter, soaking the worms in a solution of dsRNA or
simply by feeding worms with E. coli expressing dsRNA5. RNAi feeding libraries
covering most of the predicted genes in the genome are already available for use,
making screening by feeding an attractive approach. In my screen, I have made
use of the RNAi library made by the Ahringer group, which covers ~86% of
predicted genes6.
Finally, C. elegans have 5 pairs of autosomes and 1 pair of sex
chromosomes, and sex determination of the worm between hermaphrodite or male
depends on the ratio of sex chromosomes to autosomes (XX resulting in a
hermaphrodite and XO in a male). Males naturally occur in wild-type populations
but at a low frequency (~0.1% of the total population on average)7, 8. Any
mistakes in pairing and subsequent stabilization of homologous chromosomes
could lead to non-disjunction, which in turn could result in embryonic lethality
(emb) from loss of an autosome or a high incidence of males (him) from loss of
the X chromosome. This provides one way to phenotypically assess the effect of
RNAi knockdown of a gene on chromosome movement and pairing.
I.4 – Meiosis Specifics in C. elegans
In yeast and mammals, the telomeric ends of chromosomes move to
associate with the NE and they then cluster to one end of the nucleus to form a
meiotic "bouquet" for chromosome sorting9. At the NE there are SUN/KASH
complexes which span the NE and contain an inner nuclear membrane (INM)
SUN (Sad1 and UNC‑84) domain, which can bind to the nucleoplasmic domains
of other NE-bound complexes as well as to the nuclear lamina, and an outer
nuclear membrane (ONM) KASH (Klarsicht, ANC‑1 and SYNE homology)
4
domain, which can interact with motor proteins and the cytoskeleton on the
cytoplasmic side10. These SUN/KASH complexes are highly mobile during
meiosis and act to connect both the cytoplasm and the cytoskeleton network to the
nucleoplasm and to transmit forces that allow chromosome movement11. There is
evidence to suggest that the movement of chromosomes is necessary for proper
chromosome homology search, and this is true in other organisms including
C. elegans12, 13. In addition, this general mechanism of chromosome ends (with
the help of adaptor proteins) moving to contact the NE at the highly conserved
SUN/KASH proteins in order for movement and proper homology assessment to
take place is also found in C. elegans, indicating its importance across species. In
C. elegans however, it is the cis-acting PCs located near one end of each
chromosome, instead of telomeres, which associate with the NE, and the
SUN/KASH complex is named SUN-1/ZYG-12.
The role of PCs in homologous chromosome pairing and segregation was
elucidated by experiments with reciprocal translocations between nonhomologous chromosomes, deletions, and duplications. These all affected the
frequency of recombination which suggested there was a region at one end of
each chromosome that was responsible10, 14. In an experiment where both copies
of a PC were deleted, there were severe problems with chromosome
recombination and segregation10, 14, 15. Starting at the leptotene/zygotene stage, the
ZIM/HIM-8 family of zinc-finger proteins bind specifically to PCs on their
respective chromosomes in a CHK-2 kinase dependent manner: ZIM-1 binds
chromsomes II and III, ZIM-2 binds chromosome V, ZIM-3 binds chromosomes I
and IV and HIM-8 binds chromosome X16. The PLK-2 kinase, which recognizes
phosphorylated targets, is then recruited to the PC by ZIMs/HIM-8 via an
unknown mechanism2. When the PC localizes to the NE, PLK-2 becomes visible
in reporter-tagged strains at the nuclear membrane first as individual foci, then as
larger aggregates (patches), corresponding to individual and multiple, grouped
chromosome ends2 (Figure 3). The localization of PLK-2 to the NE is required for
the association of PC proteins and SUN-1 into foci2. Furthermore, PLK-2 is
required for the phosphorylation of SUN-1 at serine 12 which in turn promotes
5
aggregation of these foci into the patches required for the proper movement,
homology assessment and pairing of chromosomes2 (Figure 4). Once homologous
chromosomes have been properly sorted and their pairing stabilised by the PC,
synapsis (SC formation) is thought to initiate at the PCs15. Once initiated, the
process of synapsis is independent of homology, as largely demonstrated by
mutants defective in homolog pairing still being capable of promoting synapsis
and by translocation mutants which exhibit synapsis of all chromosome regions
despite the lack of homology15. There are also SC-deficient mutants that are still
capable of homologous associations17. It should be noted that in C. elegans and
Drosophila, synapsis is also independent of recombination, unlike in yeast, mice
and Arabidopsis17, 18.
I.5 – Cytoskeleton
The directed movements of cellular components in a cell, including the
movement of chromosomes during homology search, are largely due to the
activities of motor proteins and the cytoskeleton tracks they use19,
20
. The
cytoskeleton is the structural scaffolding of a cell and is made of up three different
protein filaments: actin microfilaments (MFs), intermediate filaments (IFs) and
microtubules (MTs).
MFs are formed from multiple G-actin subunits polymerized into F-actin
in a head-to-tail fashion. The orientation of actin results in polar MFs with the
faster growing barbed end as the plus end, and the pointed end as the minus. They
are somewhat randomly oriented throughout cell, but generally they have an
ordered structure in the cell cortex with plus-ends pointed outwards21. MFs are
generally shorter than MTs and are thought to act as local transport networks,
bridging the space between MTs, to allow uniform distribution of cargo
throughout cell21. They are used by the molecular motor myosin.
IFs come in several different forms and they can all dimerize and
self-assemble into filaments. Unlike MFs and MTs, they lack polarity and
therefore have no motor proteins associated with them22. They span the cytoplasm
and attach to MFs, MTs, and cell-cell and cell-matrix adhesion sites. A special
6
kind of IF is nuclear lamin as these are found inside the nucleus. C. elegans has
only the B-type lamin LMN-122.
MTs are formed from alternating α-tubulin and β-tubulin GTPases joined
in a head-to-tail manner to form dimer subunits. The dimers bind GTP and
polymerize to form protofilaments. Like MFs, MTs have polarity: the α-tubulin
end of one dimer forms the minus-end and the β-tubulin end of another dimer
forms the faster growing plus-end. Once a dimer has polymerized with other
dimers, the β-tubulin GTP is hydrolyzed to give GDP (α-tubulin bound GTP is
never hydrolyzed). β-tubulin GTP hydrolysis is necessary for switching between
MT catastrophe (shrinking) when GDP-bound, and MT rescue (growing) when
GTP-bound23. The newly added GTP-bound β-tubulin forms a cap at the tip of
the MT protecting it from disassembly23. These protofilaments then associate with
one another laterally to form MTs. MT organization is cell-type specific but they
are typically radially organized with the minus-ends anchored to a microtubule
organizing center (MTOC), such as the centrosome located near the nucleus, and
with the plus-ends spreading out towards the cell periphery. They are used by the
molecular motors kinesin and dynein.
I.6 – Regulation of Cytoskeleton
One way the cell controls chromosome movement is by restructuring the
cytoskeleton network via regulating polymerization and depolymerization. By
changing the direction, length and location of filaments, motor proteins can then
be tasked to move chromosomes in a very specific way, such as to form the
meiotic "bouquet" necessary for homologous chromosome pairing9.
A process known to utilise this regulated rearrangement of the
cytoskeleton is the onset of mitosis; the cell’s extensive MF network is quickly
dismantled and rearranged giving mitotic cells their characteristic round shape24.
Actin has also been shown to play an important role in forming part of the
contractile ring necessary for cytokinesis as well as in the separation of
centrosomes24. Actin nucleators and polymerases include the Arp2/3 complex
(comprised of ARX-1 to -7) and Rho GTPases (RHO-1, CDC-42, CED-10,
7
RAC-2 and MIG-2)25, 26. Arp2/3 is essential as any disruption in expression by
RNAi of Arp2/3 genes generally results in embryonic arrest25. Additionally,
WSP-1 interacts with and activates Arp2/3 and wsp-1(RNAi) worms
phenotypically resemble worms with RNAi directed against Arp2/325. The Rho
GTPases have similarly severe RNAi phenotypes ranging from axon migration
defects to emb26. Actin depolymerizering factors UNC-60A and UNC-60B are
two, tissue-specific isoforms encoded by unc-60 that differentially regulate actin
filament dynamics24, 26, 27.
The growth rate of MTs is dependent on tubulin concentration and the
association rate of tubulin to the plus-end. The dynamics of MTs are controlled in
part by nucleating factors such as γ-tubulin (TBG-1), AIR-1, SPD-2 and SPD-5,
and by plus end tracking proteins (+TIPS)23, 28, 29. The polymerizing, XMAP215
family kinesin, ZYG-9 forms a complex with TAC-1 and acts as a catalyst to
increase MT growth rate, but does not affect the rate of catastrophe28,
30
. The
doublecortin family suppresses catastrophe and leads to more stably growing
MTs30, and ZYG-8 is a doublecortin-like kinase known to affect mitotic spindle
movement28. MT depolymerases are ATP-dependent and they accelerate
shrinkage and induce catastrophe via removal of the GTP-cap30. The main ones in
C. elegans are KLP-7 (MCAK homolog), which affects both plus and minus-ends,
and KLP-3 and KLP-17 (kinesin-14 homologs), which cause depolymerization at
the plus-end despite being minus-end directed30. Other +TIPs include: MT end
binding protein (EBP-1) which affects growing tip structure; cytoplasmic linker
proteins CLIP-170 and CLIP-115, which rescue shrinking MTs and convert them
to growing MTs despite not binding directly at the site of depolymerization; and
CLIP associated proteins (CLASPs) CLS-1, CLS-2 and CLS-3, VAB-10
(spectraplakin) and APR-1 (APC-related), which coat the distal end of MTs and
act as MT stabilizing factors23,
26
. +TIPs have also been shown to attach and
stabilize MT cellular structures either by linking MT ends directly to actin
(CLASPs, spectraplakins and APC) or to cortically bound factors (CLASPs and
APC)23. Altogether the proteins can work synergistically to modulate MT
8
attachment and stabilization for targeted movement of cargo to precise cellular
sub-locations.
I.7 – Molecular Motors
While the cytoskeleton plays key roles in the control of movement,
ultimately it is the motor proteins that use the cytoskeleton “highway” to actually
confer movement to the chromosomes. Motor proteins bind to cargo with their
“tail” whilst binding to their respective cytoskeleton filament with one of their
“heads”. Forward movement arises from the coordinated binding and unbinding
of their two heads, powered by hydrolysis of ATP21. There are three classes of
motor protein: the MF motor myosin, and the MT motors kinesin and dynein.
Myosins are the only class of motors to bind MFs and have both plus and
minus-end directed myosins. Since MFs are usually organised with their plus-ends
at the cell periphery, the more common plus-end directed motors, such as
myosin-II, can transfer cargo right to the cell surface, while minus-end directed
motors, such as myosin-VI, can take internalized cargo from endocytosis to the
cell center21.
Kinesins bind MTs and most move in a plus-end directed manner towards
cell periphery. They have been shown to be very efficient transporters in vitro and
their processivity (the property of a motor to take many consecutive steps before
detachment) can be increased by having multiple kinesins bind the same cargo21.
Like the myosins, there is a family of kinesin-14 kinesins that are capable of
walking towards the opposite, minus-end direction30.
Cytoplasmic dynein also binds MTs, but they move in a minus-end
directed manner towards the cell interior. They differ from myosins and kinesins
in that they have a much more complex structure composed of two heavy chains
that contain ATPase and motor activities, two intermediate chains thought to
anchor dynein to its cargo, two light intermediate chains, and several light
chains31. They are also not as efficient at the single-motor level, as their
processivity is more variable due to their greater dependence on available ATP
and load size. However, at the cell level they are robust transporters due to their
9
ability to have multiple motors attached to the same cargo working in tandem, and
their ability to bind accessory proteins, such as dynactin, which help to enhance
their activity21.
In order for motor proteins to interact with chromosomes that are kept
isolated from the cytoplasm by the NE, the intermediary bridge SUN-1/ZYG-12
complex is needed. SUN/KASH complexes can also bind and anchor the three
types of cytoskeleton to the nuclear envelope, and these interactions can confer
movement or tension and cause force-induced changes in gene expression, a
process known as mechanotransduction22.
I.8 – Regulation of Motor Proteins
Besides regulating the cytoskeleton tracks they move along, motor
proteins themselves can be regulated and coordinated to affect intracellular
movement. As motor proteins move in a stereotypic way, it logically follows that
using different plus-end or minus-end directed motor proteins can dictate the
direction of movement of cargo. In some systems, the same cargo can move using
both MT and MF networks and can also switch motors too21. There is also the
possibility of increasing the number of motors that are attached to the same cargo
to improve processivity and, if different classes of motors bind, to allow
bidirectional movement, as is the case with nuclei during nuclear migration and
rotation32. Previous studies have shown that dynein interacts with nuclear KASH
domain proteins to generate forces used to move nuclei during nuclear migration
and rotation, centrosomes during mitosis and meiotic chromosomes to coordinate
pairing and synapsis11, 29, 32-35. There has also been evidence that dynein controls
kinesin-driven movement to achieve greater control of the kinetics of cargo
movement36-38. For example, bidirectional nuclear migrations require kinesin-1 as
the major force generator and, in a subset of migrations, dynein to regulate the
bi-directionality36. Kymographs of this migration indicated that the nucleus had
periods of no or very slow movement interrupted by periods of faster movement36.
Strikingly, similar slow/fast movement phenotypes are observed in meiotic
chromosomes at the TZ in wild-type worms (data not shown, 12, 34). This suggests
10
that a similar mechanism of action could be taking place to confer movement to
meiotic chromosomes.
Another level of regulation of motor proteins comes from accessory
proteins. Dynein has several subunits and each has a list of proteins that interact
specifically with them to, for example, help recruit the right cargo or regulate the
efficiency of motor function21. Dynactin is one such protein: it attaches to MTs
and acts as a second binding site for dynein and improves its processivity in
vivo21. Another accessory protein is the MT binding protein LIS-1, which
increases dynein activity and helps with cargo selection as well as interacts with a
subunit of dynactin31. This means that not only can the motor proteins themselves
be regulated, but the accessory proteins they associate with can be modified to
indirectly modify motor protein behaviour as well.
Finally, the many different roles that the same motor protein plays is partly
regulated by different cargo adaptors recruiting the motor protein at different
locations and at different times29,
32
. Fridolfsson et al. (2010) has shown that
UNC-83, another KASH domain protein located at the nucleus, interacts with
both dynein and kinesin-1 complexes to control nuclear migration in hyp7
embryonic hypodermal precursor cells32. In another study by Zhou et al. (2009),
they found that ZYG-12-mediated recruitment of dynein to the NE is required to
maintain MT organization, membrane architecture and nuclear positioning within
the C. elegans syncytial gonad29. They also propose a model whereby gonad
architecture is determined by MT nucleation at the plasma membrane (rather than
centrosomes) combined with tension created on the MTs by dynein anchored at
the nucleus by ZYG-12 (Figure 5)29.
I.9 – Interaction Between Different Cytoskeleton Networks
Interestingly, several studies have shown that there is a lot of
cross-regulation between MT and MF networks, particularly in relation to dynein.
In a previous study, it was found that mutations in both LIS-1 and DHC-1 (dynein
heavy chain), proteins usually thought to be required for MT-dependent events,
both disrupted the F-actin cytoskeleton in the C. elegans germline in a similar
11
manner31. They also found that treatments that alter F-actin levels or organization
(such as RNAi knockdown of actin-capping proteins) could suppress a
temperature-sensitive dhc-1(or195ts) allele and change DHC-1 localization,
implying that F-actin could also affect dynein activity by modifying its
localization. In a different study, it was found that myosin could indirectly affect
astral MT positioning by regulating partitioning protein (PAR) polarity, which in
turn regulates dynein-dynactin mediated, MF-dependent cortical forces that then
act on astral MTs to affect mitotic spindle location39. The large amount of
interactions between cytoskeleton networks that can potentially affect dynein
activity has broadened the scope of possible regulatory factors that could
indirectly affect chromosome movement.
I.10 – The Nucleoskeleton
Finally, recent work has begun on elucidating the architecture of the
nucleus (termed the “nucleoskeleton” by Tsai and McKee10) and what role this
may have on regulating the genome. The most well understood component of the
nucleoskeleton are the lamins. C. elegans only has the B-type lamin LMN-1, and
as lamins lack polarity, they do not have any associated motor proteins22. A
number of INM proteins associate with the nuclear lamina and with each other
such as LEM-2, EMR-1 and BAF-1. Together these work with the nuclear lamina
to help segregating chromosomes attach to the reassembling nucleoskeleton and
nuclear envelope22. There is evidence of actin present in the nucleus that form
unconventional polymers rather than the typical F-actin polymers, but how it is
regulated or what role it plays exactly is still unclear22. Surprisingly, myosins and
kinesins have also been discovered in the nucleus, despite a lack of conventional
track, and give weight to the idea that not all movement within the nucleus
happens by random diffusion22. In HeLa cells, nuclear MYO1C has been shown
to localize with actin and RNA polymerase I at active transcription sites and
localize in discrete puncta outside nucleoli in quiescent cells22. Though this
suggests this particular motor protein is necessary for transcription, it is not so
12
far-fetched to hypothesize that the other motor proteins found in the nucleus may
potentially have a role in chromosome dynamics in C. elegans.
I.11 – Research Proposal
Though the movement of chromosomes is critical for the proper pairing of
homologues, how the movement of chromosome ends to the NE is regulated, how
chromosome ends are attaching to the NE and how PLK-2 is recruited to the PCs
in order for homologue searching and pairing to take place remain outstanding
questions. Thus the overall goal of my project was to take advantage of the many
tools available with C. elegans to try and identify factors involved in the
chromosome movement necessary for pairing. I proposed to accomplish this by
conducting a targeted RNAi screen of candidate genes using a strain containing a
mCherry tagged version of PLK-2, which would uniquely mark all chromosome
ends simultaneously, and a GFP tagged version of H2B to visualize nuclei. I then
screened for changes in localization or movement of PLK-2 as a read-out for
chromosome end movement and localisation.
13
Chapter II: Materials and Methods
II.1 – Plates and Culture
Bacterial strains were cultured in 2×TY liquid culture medium which was
made with 16g bacto-tryptone, 10g yeast extract and 5g NaCl made to a final
volume of 1L using distilled H2O, then autoclaved and sterilised. Solid NGM
made with 20g agar, 0.55g Tris HCl, 0.24g Tris base, 3.1g bacto-peptone, 2g
NaCl and 0.008g cholesterol made to a final volume of 1L using distilled H2O,
then autoclaved and sterilised.
Clones were taken from the Ahringer C. elegans RNAi bacterial library,
along with the clone carrying the empty L4440 vector as a negative control, and
cultured overnight in 3ml of 2×TY liquid culture medium with 100μg/mL
ampicillin for selection purposes. 70µl of the RNAi bacteria were then added into
each well of a 12-well plate containing solid NGM made with a final
concentration of 50μg/mL ampicillin and 1mM IPTG, for selection and RNAi
expression, respectively. Each of the 12-well plates contained 1 negative control
well with empty L4440 vector and 11 experimental wells. Plates were left
overnight at room temperature to induce dsRNA.
II.2 – C. elegans culture
The EZ340 strain used in the screen was created by crossing EZ332
plk-2(tm1395) I; ttTi5605 vv1515 II; unc-119(ed3) III, a strain containing the
plk-2 gene tagged with mCherry, driven by the endogenous pie-1 promoter
(driving expression in the germline) and in the background of the plk-2 deletion,
with AZ212 unc-119(ed3) ruIs32 III, a strain containing the histone H2B tagged
with GFP also driven by pie-1.
Worms were synchronized and sterilized via sodium hypochlorite (bleach)
treatment. Worms and eggs grown at 20C were rinsed off NGM plates with
distilled water into a conical tube. An equal volume of 2×bleach solution was
added to the tube and the tube was mixed then vortexed every 2min for 7min to
ensure all the worms had been lysed. Using sterile techniques, more distilled
14
water was then added till full and the tube shaken before being spun down for 40s
at 1900RPM. The water was decanted leaving just the pellet of eggs. This wash
procedure was repeated another two times to remove as much bleach as possible.
After the last wash, M9 solution was added to the pellet of eggs and the tube was
left overnight at 20C on a rocker to allow all eggs to hatch and to arrest at L1
diapause stage.
II.3 – Feeding RNAi and Scoring
Identical volumes of L1 worms (~15 – 20 worms) were pipetted into each
RNAi well. Worms were allowed to develop into adults on the RNAi over 3 days
at 20C before being observed under the microscope. For the qualitative
assessment of emb and him conducted on the positive candidates, on the fourth
day of feeding, having laid their eggs, the adults were then removed from the
wells. The following day, the number of dead eggs was qualitatively scored by
eye as compared to wild-type. Worms were considered as emb if the number of
dead eggs was greater than wild-type by 10 or more eggs. Two days later, the
number of males was also qualitatively scored by eye as compared to wild-type.
Worms were considered as him if there were 3 or more males than wild-type.
II.4 – Time-lapse Microscopy
Live, young adult worms were removed from the RNAi wells after 3 days
of feeding and mounted on 2% agarose pads in 3mM levamisole (acting as a mild
paralyzing agent) and covered with a coverslip sealed with nail polish. TZ nuclei,
marked by H2B::GFP as having a crescent-shape due to chromosome clustering,
were observed at room temperature for the localization and movement of
PLK-2::mCherry using a DeltaVision RT system (Applied Precision) on an
Olympus IX70 inverted microscope, equipped with 100× or 60× oil immersion
objective NA 1.35 U Plan Apo (Olympus) and a Cool SNAP HQ/ICX285 CCD
camera. 3min movies of TZ nuclei were assembled from time-lapse images
acquired every 10s in stacks of 6 optical sections with 0.5µm increments, with 1s
exposure time. Acquisition and de-convolution of images was performed using
15
standard parameters in softWoRx software (Applied Precision). Movement was
determined subjectively by observing the movies for noticeable changes in
localisation.
II.5 – Immunostaining
Worms were fed using the previous RNAi protocol. On the third day of
feeding however, instead of being observed under the microscope, worms were
removed from the plate and placed in a drop of 1×PBS on a microscope slide
coated in poly-lysine. Gonads were then dissected out from the worms and fixed
in 1% PFA for 5min before being freeze-cracked in liquid nitrogen and dipped in
cold methanol for 1min. Slides were then washed with 1×PBST 3 times, 5min
each time, followed by a blocking period in 1% BSA in PBST for 1h. Primary
antibody anti-HTP-3 (1:100) was then added and left overnight at 4ºC. Slides
were then washed in 1% BSA in PBST 4 times, 10min each time. Secondary
antibody Alexa 488 (1:1000) was then added and left for 2h at room temperature.
The slide was then washed again in 1% BSA in PBST 4 times, 10min each time,
before staining with 13µl of 1µl/mL DAPI in Vectashield and sealed with a
coverslip and nail polish.
16
Chapter III: Results
To attempt to answer the question of what factors are involved in chromosome
movement during prophase I of meiosis, I conducted a candidate-approach RNAi
screen to try and identify potential candidates by observing worms to see if there
were any defects in PLK-2 localisation or movement.
III.1 – Selecting RNAi Candidates
Due to the time-intensive nature of the screening methodology, I opted to
take a candidate approach to the screen instead of a genome-wide analysis.
Candidate genes were selected from lists of MT regulators40, 41 and cytoskeleton
related proteins, such as kinesins, as the cytoskeleton and its components make up
a critical component of meiotic chromosome movement and homology search.
Genes were also selected from a list of all kinases found in C. elegans42 since
PLK-2 recognizes phosphorylated targets. Finally, genes were selected from a
small subset of chromatin related genes and candidates previously pulled down in
two immunoprecipitation (IP) experiments, one using HIM-3 (High Incidence of
Males 3) and another using HTP-3 (Him-Three Paralog 3). HIM-3 is a
meiosis-specific component of chromosome cores that is essential for synapsis
and chiasmata formation, and him-3 mutants show defects in meiotic chromosome
segregation43. HTP-3 is another chromosome axis protein which was found to
form complexes with HIM-3 as well as double-strand break repair components,
and furthermore has been shown to be required for proper homologue alignment,
synapsis and crossing over44,
45
. As such, the candidates from the HIM-3 and
HTP-3 IP experiments are attractive candidates in the search for factors involved
in chromosome movement. The compiled list of candidates was then checked
against a list of genes available from the Ahringer C. elegans RNAi feeding
library to generate a final list of candidates readily available for testing (Table 1).
Those genes that were potential candidates but were not tested due to their
unavailability in the Ahringer library are found in Table 7.
17
III.2 – Controls
To ensure that the lack of movement phenotype was not observed in
wild-type situations, transition zone nuclei of untreated, wild-type EZ340 worms
and EZ340 worms fed control RNAi of the empty L4440 vector were observed
and compared. In both cases, PLK-2 was seen to localize to the nuclear periphery
in dynamic foci and patches, corresponding to single or multiple chromosome
ends (Figure 6A and Figure 7A). There were also no observable defects in gonad
morphology nor emb or him phenotypes in the either the untreated or RNAi-fed
worms.
CHK-2 is a serine/threonine kinase that is required for nuclear
reorganization and homolog interactions, and that also has been shown to be
required for proper PLK-2 localisation to the NE2, 46. To ensure that RNAi feeding
is capable of knocking down kinase function in the gonad and to ensure that
changes in PLK-2 localization can be detected, chk-2 RNAi was conducted. This
resulted in a lack of foci or patches at the nuclear periphery as compared to
wild-type (Figure 6), confirming RNAi feeding’s potential efficacy in the gonad.
III.3 – Results of RNAi Screen
The targeted RNAi screen positively identified a total of 156 out of 482
genes for which RNAi causes a lack of PLK-2 movement in young adult
germlines (Figure 9 and Table 2). Of the 156 positive candidates, 118/377 were
kinases, 15/32 were cytoskeleton-related, 11/28 were chromatin-related, 7/19
were from the HTP-3 IP experiment, 3/8 were from the HIM-3 IP experiment and
2/18 were MT regulator genes. Positive candidates displayed normal PLK-2
localisation to the nuclear membrane and formed both foci and patches, but
foci/patch movement was not as dynamic as in wild-type, and seemed, by
subjective measure, to have been completely halted or severely slowed as seen in
Figure 7.
I attempted to further characterize some of the positive candidates by
checking for emb and him phenotypes as indicators of improper chromosome
18
movement and/or pairing. Of the 156 genes positively identified, 24 have also
given positive hits for emb (Table 3) and 1 gene was positive for him (Table 4).
19
Chapter IV: Discussion and Future Directions
IV.1 – Candidate Gene syp-2 Validates RNAi Screen
Methodology
One of the positive candidates identified in the screen with an emb
phenotype was syp-2. SYP-2 is a structural component of the SC and is required
for maintaining stable pairing between homologous chromosomes during
meiosis17. Germlines from young adult worms fed syp-2 RNAi were dissected and
doubly stained with DAPI and HTP-3 antibody to visualize DNA and any defects
in synapsis during the later stages of meiosis. 12 univalents were clearly present
in the oocytes suggesting problems in synapsis (Figure 8).
Previous work done by Baudrimont et al. (2010) has shown that mutants
defective in SC formation have reduced movement of SUN-1 aggregates34. As
PLK-2 has been shown to co-localise with SUN-12, the SUN-1 aggregates could
also be used to infer the position of chromosomal attachment plaques, and
subsequently chromosome end movement. This impairment of chromosome end
movement suggests that defects in SC formation may hinder movement, perhaps
by affecting functional attachments to SUN-1/ZYG-12 complexes34. Importantly,
the reduced movement of SUN-1 previously noted corroborates the validity of the
loss of movement phenotype observed from syp-2 RNAi, and confirms that the
methodology employed is, in fact, capable of detecting genes affecting
chromosome movement as predicted.
IV.2 – emb Phenotype Not Necessarily Linked to Lack of
Chromosome Movement Phenotype
In some instances, foci/patch movement of PLK-2 seemed as wild-type in
some worms, despite having an overall emb phenotype (Table 5). While this may
be due to incomplete knockdown of gene expression by feeding, it is more likely
to indicate that other factors, besides those affecting chromosome movement or
20
egg production, may be causing emb, such as defects in somatic cytoskeleton
regulation.
Similarly, lack of PLK-2 movement does not always result in immediate
emb or him phenotypes. This could be explained once again by incomplete
penetrance of the RNAi, but it may also be explained by possible non-autonomous
effects that cause a disruption in reproduction, such as conditions of stress.
Previous studies have shown that environmental stress can affect brood size and
reproduction, even post L1 diapause or dauer (an alternate larval stage that is
resistant to unfavourable conditions). In one study by McMullen et al. (2012),
worms were shifted from the commonly used cultivation temperature of 20ºC, just
prior to the onset of reproduction, to various elevated temperatures, and clear
differences were seen in the number of eggs laid47. In another study by Dalfó et al.
(2012), it was observed that sensory cues reporting population density and food
abundance affect the balance of proliferation versus differentiation in the germline
via TGF-β48. Stress, or improper signalling of stress conditions due to RNAi
effects, could therefore cause disruption in reproductive progression, as seen by
unmoving chromosome ends.
Furthermore, as subsequent generations were not examined for fitness
(e.g. brood size, life span, physical defects), candidate genes that gave rise to a
lack of PLK-2 movement without emb or him in the progeny may have had a less
severe and obvious effect on chromosome pairing and movement. Defects may
have therefore only appeared during the later stages of development in the
progeny, and as such, were undetected by the screen.
IV.3 – Future Directions
Since the goal of my project was to try and identify factors involved in
chromosome movement, there are many potential avenues for further
characterization and study. I have outlined and discussed some of these avenues
below.
As the RNAi screen was conducted using a candidate-approach, it would
be interesting to expand the scope of the screen to include the missing kinases, all
21
the phosphatases or, time and resources permitting, the entirety of the rest of the
C. elegans genome. In this way, a clearer picture of all the potential genes
involved in chromosome movement during meiosis would be gained.
Furthermore, by expanding the pool of sample genes, genes may be found that
show striking phenotypes heretofore unobserved, similar to the prominent change
in PLK-2 localization found in chk-2 RNAi worms. An example of one such
phenotype would be a change in the relative number of foci or patches present at
the nuclear periphery, where a greater number of foci might suggest a problem in
proper homologue recognition and pairing and where a greater number of patches
might suggest a problem in properly pulling apart non-homologous chromosomes
for sorting.
It would also be worthwhile to further study the candidates involved in
molecular motor activity (Table 6). As dynein has been shown to both be required
for proper chromosome movement and to work in tandem with kinesin-1 to
regulate bidirectional movement of nuclei, it would be interesting to see if a
similar mechanism is in place to give rise to the highly dynamic nature of
chromosomes during homology search. However, as it is notoriously difficult to
disrupt motor activity without affecting vital processes, care will have to be given
in designing informative experiments. One possibility would be to test abolishing
dynein by using a fast-acting, temperature sensitive dynein mutant49. Once the
animals have been shifted to the restrictive temperature, the TZ nuclei could be
observed using live-imaging to record any changes in chromosome movement
over time. This would reduce the number of non-autonomous effects arising
simply from cytoskeletal defects accumulated over longer periods of dynein
inhibition. Similarly, kinesins could be specifically targeted during adulthood
using kinesin-specific drugs50 to try and avoid knocking out vital kinesin function.
Another important step to carry out would be to test the positive
candidates further by looking for other signs of meiotic defects. DAPI staining
could be used to quickly assess gross morphological defects, such as an extended
TZ (indicating potential delay or defects in resolving homologous pairs) or
univalents in oocytes. Another test would be to look for defects in proper pairing
22
between homologs. Chromosome specific markers, such as HIM-8 to mark X
chromosome PCs51 or ZIM-2 to mark V chromsome PCs16, could be used to see if
homologous chromosomes have properly paired or not (1 signal versus 2
respectively) despite defects in movement. Examining brood size as compared to
wild-type would also be another method of assessing meiotic defects in addition
to looking to see if there are emb and him phenotypes. Finally, conducting
generational studies on the positive candidates would help to better characterize
the effects of each candidate gene on the integrity of the genome over time.
23
Chapter V: Conclusion
Chromosome movement resulting in proper homologous pairing is controlled and
regulated in a manner that is not well understood. To try and identify factors
involved in this process a targeted RNAi screen was conducted. 482 candidates
were selected for testing from a list of 377 kinases, 28 chromatin-related genes, 32
cytoskeleton-related genes, and genes pulled down in two different IP
experiments using HIM-3 and HTP-3 (8 and 19 respectively). Of these 482 genes,
156 were positively identified as having a lack of PLK-2 movement in TZ nuclei
in young adult germlines, suggesting that they may play a role in chromosome
movement. 118 were kinases, 15 were cytoskeleton-related, 11 were chromatinrelated, 7 were from the HTP-3 IP experiment, 3 were from the HIM-3 IP
experiment and 2 were MT regulator genes. 24 of these positive candidates were
also positive for emb: 7 were kinases, 5 were cytoskeleton-related, 6 were
chromatin-related, 2 were from the HTP-3 IP experiment, 2 were from the HIM-3
IP experiment and 1 was a MT regulator gene. 1 gene from the HTP-3 IP
experiment (him-1) was additionally positive for him. Since positive candidates
were found in each of the gene classes tested, this suggests that the process of
controlling and regulating chromosome movement during homology search is
multi-factorial. These candidates also present many possible directions for future
projects for further validation and characterization.
24
Figures
Figure 1 Phases of meiosis
Graphical representation of key chromosome movements during meiosis. One
pair of homologous chromosomes is shown in red and pink lines, whereas pairs of
sister chromatids are shown in the same colour. (A) Steps in prophase I. (B) How
paired, homologous chromosomes are eventually segregated into separate
chromatids. Taken from Tsai and McKee (2011)10
25
Figure 2 Diagram illustrating gonad structure
Cartoon representation of one arm of the C. elegans gonad. Meiosis progresses
proximally from the distal tip. TZ nuclei are crescent shaped. Modified from
Bickel et al. (2010)52
26
Figure 3 Chromosome end attachment to nuclear envelope
Diagram illustrating chromosome ends with PC proteins and PLK-2 recruited to
the nuclear envelope SUN-1/ZYG-12 complex, which in turn is connected to the
cytoskeleton via motor proteins. Modified from Jaspersen and Hawley (2011)53
27
Figure 4 Progression of chromosome movement and pairing
At the onset of meiosis, chromosome ends associated with the PCs attach to the
NE. SUN-1/ZYG-12 links the chromosome ends to cytoskeleton forces.
Chromosome movement through these linkages brings variable numbers of
chromosomes into larger SUN-1/ZYG-12 patches in which homology between
pairs of chromosomes is assessed. Non-homologous chromosomes are separated
while homologous chromosomes initiate synapsis at the PC. Taken from Labella
et al. (2011)2
28
Figure 5 Proposed model for role of microtubules in regulating nucleus
positioning
Physical linkages between the cytoskeleton and the nucleus via trans-membrane
proteins and motor proteins could be used to both position the nucleus and move
chromosomes during meiosis. Taken from Zhou et al. (2009)29
29
Figure 6 chk-2 RNAi causes loss of PLK-2 foci and patches
TZ nuclei of EZ340 worms are indicated by white brackets. (A) Wild-type
localisation of PLK-2 as foci and patches at the nuclear periphery. (B) chk-2
RNAi fed worms lack PLK-2 localisation as foci or patches.
30
Figure 7 Comparison of WT vs no movement phenotypes
Time-lapse microscopy of TZ nuclei of EZ340 worms. Arrows highlight nuclei
that exemplify respective movement phenotypes. White boxes denote magnified
nuclei. Both PLK-2::mCherry foci and patch movement were reduced with RNAi
feeding of osm-1 (B) as compared to wild-type (A). Slight movements of PLK-2
foci/patches seen in B are due to movement of the worm during live-imaging.
31
Figure 8 syp-2 RNAi results in clear defects in the gonad
Gonad from young adult worm fed syp-2 RNAi. DAPI-stained DNA is in blue,
HTP-3 is in green. 12 univalents are present in late stage oocytes as shown in
magnified insert. This is due to problems in SC formation leading to problems
with proper synapsis.
32
A
B
118
326
15
156
11
7
3
2
Kinases
Cytoskeleton-related
Chromatin-related
HTP-3 IP
HIM-3 IP
MT Regulators
Figure 9 Break down of positive candidates
Figure illustrating break down of positive candidates. (A) 156/482 genes were
positively identified (in green). (B) Of the 156 positive candidates, there were
118/377 kinases, 15/32 cytoskeleton-related, 11/28 chromatin-related, 7/19 HTP-3
IP, 3/8 HIM-3 IP and 2/18 MT regulator genes.
33
Tables
Table 1 List of all candidate genes
Gene
Sequence
Chromatin-related
lin-59
met-1
cogc-5
pop-1
taf-4
cdc-6
dapk-1 (mor-3)
mes-3
lin-61
hil-8
lin-35
C06A5.3
slx-1 (giyd-1)
B0261.1
let-526 (C01G8.7)
let-526 (C01G8.8)
smo-1
hmg-3
ekl-1
swsn-9 (tag-298)
C01H6.9
T23H2.3
chd-1
rskn-2
pbrm-1
dcp-66
taf-11.2
AC8.6
T12F5.4
C43E11.3
C43E11.11
W10C8.2
R119.6
C43E11.10
K12C11.4
F54C1.3
R06C7.7
T05E8.2
C32F10.2
C06A5.3
F56A3.2
B0261.1
C01G8.7
C01G8.8
K12C11.2
C32F10.5
F22D6.6
C01H6.7
C01H6.9
T23H2.3
H06O01.2
C54G4.1
C26C6.1
C26C6.5
K10D3.3
AC8.6
Chr.
Gene
Sequence
HTP-3 IP (cont.)
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
X
mlh-1
hsp-1
F36D3.5
mre-11
rad-50
hcp-2
egrh-1
F28B3.7
C12C8.1
F56A3.4
F25H2.13
Y63D3A.5
W08F4.8
C18H2.1
R07E5.8
F57B9.2
F11H8.4
F59A2.1
C36A4.8
III
IV
V
V
V
V
X
HIM-3 IP
aspm-1 (tag-255)
ZK930.1
hmg-12 (hmg-I-beta)
met-2
rsa-2
R08C7.4
cap-1 (capz/a)
ZC317.1
C45G3.1
ZK930.1
Y17G7A.1
R05D3.11
Y48A6B.11
R08C7.4
D2024.6
ZC317.1
I
II
II
III
III
IV
IV
V
Cytoskeleton-related
kca-1
dyf-1
klp-15
klp-16
unc-104 (klp-1)
klp-3
lin-5
nph-1
klp-17
ddl-3
klp-6
kap-1
unc-116 (khc-1)
klp-7
klp-19
dyf-2
dyf-3
osm-3 (klp-2; caf-1)
zen-4 (klp-9)
klp-18
klp-10
klp-11
epi-1 (egf-3; lgx-3)
klp-12
HTP-3 IP
him-1 (smc-1)
hsp-70
spd-5
bch-1 (rtel-1)
tfg-1
cdc-37
C18H2.1
cku-80
let-711 (ntl-1; spn-3)
cyk-1
npp-9
brc-1
T28A8.7
F26D10.3
F36D3.5
ZC302.1
T04H1.4
T06E4.1
C27C12.2
Chr.
I
I
I
I
I
II
III
III
III
III
III
III
34
C10H11.10
F54C1.5
M01E11.6
C41G7.2
C52E12.2
T09A5.2
T09A5.10
M28.7
W02B12.7
Y54G11A.8
R144.1
F08F8.3
R05D3.7
K11D9.1
Y43F4B.6
ZK520.3
C04C3.5
M02B7.3
M03D4.1
C06G3.2
C33H5.4
F20C5.2
K08C7.3
T01G1.1
I
I
I
I
II
II
II
II
II
II
III
III
III
III
III
III
IV
IV
IV
IV
IV
IV
IV
IV
Gene
Sequence Chr.
Cytoskeleton-related (cont.)
arx-2 (arp-2)
vab-8 (klp-5; unc-107)
klp-14 (bmk-1)
klp-8
klp-4
lfi-1
klp-13
osm-1
vpr-1
apr-1
mei-1
spd-2
vab-10
dhc-1
zyg-9
tac-1 (2P40)
ptl-1 (tau-1)
unc-83 (cegrip;gip-1)
cls-2
tbg-1 (tubg; sas-3)
lis-1 (pnm-1)
unc-33
bicd-1
figl-1
spas-1
tektin
vhl-1
K07C5.1
K12F2.2
F23B12.8
C15C7.2
F56E3.3
ZC8.4
F22F4.3
T27B1.1
F33D11.11
K04G2.8
T01G9.5
F32H2.3
ZK1151.1
T21E12.4
F22B5.7
Y54E2A.3
F42G9.9
H04J21.3
R107.6
F58A4.8
T03F6.5
Y37E11C.1
C43G2.2
F32D1.1
C24B5.2
R02E12.4
F08G12.4
Gene
V
V
V
X
X
X
X
X
I
I
I
I
I
I
II
II
III
III
III
III
III
IV
IV
V
V
X
X
T09B4.7
F37E3.3
C55B7.10
nekl-2 (phi-35)
ZC581.2
ZC581.7
ZC581.9
E02D9.1
T21G5.1
smg-1 (mab-1)
T10B11.2
kin-14
prpf-4 (prp-4)
unc-14
hpo-11
F52B5.2
cdk-8
gska-3
dyf-5
F26E4.5
mom-4
Y106G6A.1
Y106G6D.4
Y106G6E.1
csnk-1
wts-1 (tag-181)
tag-344
C34B2.3
cdk-9
dlk-1
src-2 (kin-22)
F46F5.2
gcy-21
gcy-15
gcy-19
F53C3.1
C16A11.3
vab-1
tbck-1
nsy-1 (esp-8; ask-1)
F09C12.2
C34F11.5
ZK622.1
gcy-12
pkc-3
pink-1
F12A10.2
Kinases
spe-8
ZC123.4
gcy-17 (gcy-24)
F23C8.7
F23C8.8
mtk-1
cdk-2
W09C3.1
let-502
F21F3.2
W03G9.5
ppk-1
hil-8
C09D4.3
F59A3.8
bub-1
C34G6.5
B0207.7
vps-34 (let-512)
F53G12.6
ZC123.4
W03F11.2
F23C8.7
F23C8.8
B0414.7
K03E5.3
W09C3.1
C10H11.9
F21F3.2
W03G9.5
F55A12.3
T05E8.2
C09D4.3
F59A3.8
R06C7.8
C34G6.5
B0207.7
B0025.1
Sequence
Kinases (cont.)
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
35
T09B4.7
F37E3.3
C55B7.10
ZC581.1
ZC581.2
ZC581.7
ZC581.9
E02D9.1
T21G5.1
C48B6.6
T10B11.2
F22D6.1
F22D6.5
K10D3.2
H37N21.1
F52B5.2
F39H11.3
C36B1.10
M04C9.5
F26E4.5
F52F12.3
Y106G6A.1
Y106G6D.4
Y106G6E.1
Y106G6E.6
T20F10.1
B0511.4
C34B2.3
H25P06.2a
F33E2.2
F49B2.5
F46F5.2
F22E5.3
ZC239.7
C17F4.6
F53C3.1
C16A11.3
M03A1.1
C33F10.2
F59A6.1
F09C12.2
C34F11.5
ZK622.1
F08B1.2
F09E5.1
EEED8.9
F12A10.2
Chr.
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
Gene
Sequence
Kinases (cont.)
ZK177.2
C17C3.11
zyg-1
F59E12.3
C25H3.1
vrk-1 (tag-223)
C29H12.5
sma-6
dgk-5
C56C10.6
F54H5.2
T19D12.5
F41G3.5
B0252.1
B0495.2
ire-1
T05C12.1
F35C11.3
sphk-1
K08F8.1
let-23 (kin-7)
cam-1 (kin-8)
kin-15
M176.9
R09D1.12
gcy-3
gcy-2
gcy-1
wee-1.1
wee-1.3
old-2 (tkr-2; kin-28)
old-1 (tkr-1)
C08H9.8
R05H5.4
gcy-4
gcy-5
mnk-1
C14A4.13
R03D7.5
W02B12.12
age-1 (daf-23)
ZK930.1
F49C5.4
gcn-2
Y39G8C.2
madf-5
daf-19 (daf-24)
ZK177.2
C17C3.11
F59E12.2
F59E12.3
C25H3.1
F28B12.3
C29H12.5
C32D5.2
K06A1.6
C56C10.6
F54H5.2
T19D12.5
F41G3.5
B0252.1
B0495.2
C41C4.4
T05C12.1
F35C11.3
C34C6.5
K08F8.1
ZK1067.1
C01G6.8
M176.6
M176.9
R09D1.12
R134.1
R134.2
AH6.1
F35H8.7
Y53C12A.1
ZK938.5
C08H9.5
C08H9.8
R05H5.4
ZK970.5
ZK970.6
R166.5
C14A4.13
R03D7.5
W02B12.12
B0334.8
ZK930.1
F49C5.4
Y81G3A.3
Y39G8C.2
C01G12.1
F33H1.1
Chr.
Gene name
Sequence
Kinases (cont.)
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
pat-4
ver-2
grk-2 (tag-161)
C24A1.3
W04B5.5
prk-2
kin-19
C03C10.2
mpk-1 (sur-1)
cdtl-7
ckb-1
ckb-2
ckb-3
C28A5.6
F25F2.1
F26A1.3
F26A1.4
coq-8
C26E6.1
C45G9.1
T15B12.2
W03A5.1
D1044.1
pqn-25
daf-4
ckk-1
C05D10.2
kin-18 (sulu)
efk-1
gar-2 (acm-2)
zak-1
F57B9.8
F31E3.2
prk-1
R151.4
K06H7.1
K06H7.8
kin-31
pdhk-2
aak-1
ikke-1
ZK507.1
dgk-3
tlk-1
cdk-1 (ncc-1)
riok-3
D2045.5
36
C29F9.7
T17A3.8
W02B3.2
C24A1.3
W04B5.5
F45H7.4
C03C10.1
C03C10.2
F43C1.2
B0285.1
B0285.8
B0285.9
B0285.10
C28A5.6
F25F2.1
F26A1.3
F26A1.4
C35D10.4
C26E6.1
C45G9.1
T15B12.2
W03A5.1
D1044.1
D1044.3
C05D2.1
C05H8.1
C05D10.2
T17E9.1
F42A10.4
F47D12.1
R13F6.6
F57B9.8
F31E3.2
C06E8.3
R151.4
K06H7.1
K06H7.8
B0523.1
ZK370.5
PAR2.3
R107.4
ZK507.1
F54G8.2
C07A9.3
T05G5.3
ZK632.3
D2045.5
Chr.
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
Gene
Sequence
Kinases (cont.)
D2045.7
strd-1
col-94
cdk-5
W06F12.3
hcp-3
tpa-1 (nipi-1)
daf-1
F18F11.4
kgb-1
pig-1
C18H7.4
K11H12.9
mak-2
cmk-1 (Ce-CaM-KI)
F52C12.2
gcy-23
kgb-2
T22B11.3
R11E3.1
H06H21.8
C09B9.4
R13H9.5
R13H9.6
W03F8.2
ttbk-2
F36H12.9
T11F8.4
C39H7.1
C55C3.4
K08B4.5
C25A8.5
D2024.1
unc-82
plk-3
T06C10.3
kin-26
gcy-8
pmk-2
pmk-3
B0218.5
pct-1 (pct)
pkg-2
C49C8.1
F08B4.3
wnk-1
Y11D7A.8
D2045.7
Y52D3.1
W05B2.1
T27E9.3
W06F12.3
F58A4.3
B0545.1
F29C4.1
F18F11.4
T07A9.3
W03G1.6
C18H7.4
K11H12.9
C44C8.6
K07A9.2
F52C12.2
T26C12.4
ZC416.4
T22B11.3
R11E3.1
H06H21.8
C09B9.4
R13H9.5
R13H9.6
W03F8.2
F36H12.8
F36H12.9
T11F8.4
C39H7.1
C55C3.4
K08B4.5
C25A8.5
D2024.1
B0496.3
F55G1.8
T06C10.3
T06C10.6
C49H3.1
F42G8.3
F42G8.4
B0218.5
C07G1.3
C09G4.2
C49C8.1
F08B4.3
C46C2.1
Y11D7A.8
Chr.
Gene
III
III
III
III
III
III
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
Sequence
Kinases (cont.)
F49C12.7
cka-1
kin-21
kin-24
F32B6.4
kin-5
F44D12.11
frk-1
W01B6.2
W01B6.5
C04G2.2
C04G2.10
mpk-2
Y43C5B.2
F01D4.3
T25B9.4
T25B9.5
ZK593.9
M7.7
C08F8.6
C05C12.1
F22B3.8
kin-4
dyf-18
C27D8.1
gcy-18 (gcy-26)
mbk-2
pik-1
K09B11.5
Y38H8A.3
Y38H8A.4
C49C3.2
C49C3.10
gcy-27
F11E6.8
bbs-8
kin-34
nhr-83
F48G7.12
K09C6.7
K09C6.8
C38C3.4
kin-30
nhr-246
gcy-20
Y59A8A.b
Y38H6C.20
37
F49C12.7
C28D4.2
W08D2.8
K07F5.4
F32B6.4
T13H10.1
F44D12.11
T04B2.2
W01B6.2
W01B6.5
C04G2.2
C04G2.10
C04G6.1
Y43C5B.2
F01D4.3
T25B9.4
T25B9.5
ZK593.9
M7.7
C08F8.6
C05C12.1
F22B3.8
C10C6.1
H01G02.2
C27D8.1
ZK896.8
F49E11.1
K09B11.1
K09B11.5
Y38H8A.3
Y38H8A.4
C49C3.2
C49C3.10
C06A12.4
F11E6.8
T25F10.5
R02C2.2
F48G7.3
F48G7.12
K09C6.7
K09C6.8
C38C3.4
M01B2.1
ZK1037.4
F21H7.9
Y59A8A.b
Y38H6C.20
Chr.
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
V
V
V
V
V
V
V
V
V
V
V
V
Gene
gcy-22
T08D2.7
chk-1
ckb-4
F56A4.5
F59B1.8
F12F3.2
mrck-1
scd-2
gck-2
F09G2.1
C18B10.6
F20D6.5
air-1
rol-3
F38E1.3
gcy-7
gck-1
E02C12.6
E02C12.7
E02C12.8
E02C12.9
E02C12.10
E02C12.11
C50F4.1
C50F4.10
atl-1
C50H2.7
gcy-6
F32D8.10
F58B4.5
pak-2
daf-11
D2023.6
pkc-1
rskd-1
gcy-13
R90.1
T16G1.3
T16G1.4
T16G1.5
T16G1.6
T16G1.7
W07G4.3
R10D12.10
T08G5.2
Sequence
Kinases (cont.)
T03D8.5
T08D2.7
Y39H10A_
224.a
F22F7.5
F56A4.g
F59B1.8
F12F3.2
K08B12.5
T10H9.2
ZC404.9
F09G2.1
C18B10.6
F20D6.5
K07C11.2
C16D9.2
F38E1.3
F52E1.4
T19A5.2
E02C12.6
E02C12.7
E02C12.8
E02C12.9
E02C12.10
E02C12.11
C50F4.1
C50F4.10
T06E4.3
C50H2.7
B0024.6
F32D8.10
F58B4.5
C45B11.1
B0240.3
D2023.6
F57F5.5
F55C5.7
F23H12.6
R90.1
T16G1.3
T16G1.4
T16G1.5
T16G1.6
T16G1.7
W07G4.3
R10D12.10
T08G5.2
Chr.
Gene
V
V
par-1
tag-191
T01G5.1
fkh-2
ceh-93
F28C10.3
pifk-1
K02E10.7
ddr-2
dhs-27
ckc-1
sax-1
C01C4.3
F47F2.1
lin-18
hpk-1
cka-2
cst-1
dgk-2
T07F12.4
ZC449.3
F35C8.1
F35C8.2
ddr-1
tag-257
pak-1
C03B1.5
svh-2
C36B7.1
C55B6.5
mek-1
sek-1
F22F1.2
F13B9.4
kin-9
trk-1
grk-1
mkk-4
VZC374L.1
pkn-1
ZC504.3
mig-15
F59F5.3
abl-1
ver-3
ver-4
egl-15
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
38
Sequence
Kinases (cont.)
H39E23.1
C53A5.4
T01G5.1
T14G12.4
R04A9.5
F28C10.3
F35H12.4
K02E10.7
F11D5.3
C04F6.5
T27A10.3
R11G1.4
C01C4.3
F47F2.1
C16B8.1
F20B6.8
C52B9.1
F14H12.4
F46H6.2
T07F12.4
ZC449.3
F35C8.1
F35C8.2
C25F6.4
F46G11.3
C09B8.7
C03B1.5
T14E8.1
C36B7.1
C55B6.5
K08A8.1
R03G5.2
F22F1.2
F13B9.4
F08F1.1
D1073.1
F19C6.1
F42G10.2
VZC374L.1
F46F6.2
ZC504.3
ZC504.4
F59F5.3
M79.1
F59F3.1
F59F3.5
F58A3.2
Chr.
V
V
V
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Gene
pek-1
C44C10.7
mes-1
B0198.3
T01H10.4
nekl-3
T24D5.4
ppk-3
F09A5.2
K08H2.5
C29F7.2
sad-1
odr-1
gck-4
F16B12.5
K04C1.5
E02H4.3
E02H4.6
C44H4.6
mbk-1
piki-1
kin-20
nipi-3
F38E9.5
F22H10.1
F22H10.5
aak-2
gcy-11
Sequence
Kinases (cont.)
Chr.
F46C3.1
C44C10.7
F54F7.5
B0198.3
T01H10.4
F19H6.1
T24D5.4
VF11C1L.1
F09A5.2
K08H2.5
C29F7.2
F15A2.6
R01E6.1
C04A11.3
F16B12.5
K04C1.5
E02H4.3
E02H4.6
C44H4.6
T04C10.1
F39B1.1
F46F2.2
K09A9.1
F38E9.5
F22H10.1
F22H10.5
T01C8.1
C30G4.3
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Synaptonemal-Complex-Related
syp-2
C24G6.1
V
39
Table 2 List of positive candidates
Gene
Sequence
Brief Description26
Chr.
Chromatin-related
taf-4
R119.6
I
Part of the TFIID mRNA transcription complex
Homolog of origin complex component which
controls start of DNA replication
Member of a Polycomb-like chromatin repressive
complex with MES-2 and MES-6
Rb ortholog, regulates vuval development and
atagonizes Ras signaling
Component of SWI/SNF complex
Ortholog of SUMO, a ubiquitin-like moiety
No description available
Enhancer of KSR-1 lethality
Component of SWI/SNF complex
NuRD ortholog , required for excretory cell
differentiation and vulval development
Component of transcriptional regulatory
complex, required for genome stability and
postembryonic developmental processes
cdc-6
C43E11.10
I
mes-3
F54C1.3
I
lin-35
C32F10.2
I
let-526 (C01G8.8)
smo-1
hmg-3
ekl-1
swsn-9 (tag-298)
C01G8.8
K12C11.2
C32F10.5
F22D6.6
C01H6.7
I
I
I
I
I
dcp-66
C26C6.5
I
lin-61
R06C7.7
I
him-1 (smc-1)
C18H2.1
cku-80
brc-1
rad-50
F36D3.5
egrh-1
F28B3.7
C18H2.1
R07E5.8
C36A4.8
T04H1.4
F36D3.5
C27C12.2
I
III
III
III
V
V
X
HTP-3 IP
Structural maintenance of chromosomes
Protein with BRCT, WSN, and ankyrin repeats
DNA repair and resistance to IR in somatic cells
Double-strand break repair
DNA repair protein
Protein with BRCT, WSN, and ankyrin repeats
Oocyte meiotic maturation and ovulation
HIM-3 IP
aspm-1 (tag-255)
cap-1 (capz/a)
ZC317.1
kca-1
dhc-1
unc-104 (klp-1)
lin-5
klp-17
ddl-3
klp-6
klp-7
Regulates meiotic spindle organization,
movement, localization
D2024.6
IV
F-actin capping protein alpha subunit
ZC317.1
V
ATP-dependent RNA helicase
Cytoskeleton-related
Kinesin cargo adaptor, involved with meiotic
C10H11.10
I
spindle movement
Cytoplasmic dynein heavy chain homolog
required in one-cell embryos for pronuclear
T21E12.4
I
migration, centrosome separation, centrosome
proximity to the male pronucleus, and mitotic
spindle orientation
C52E12.2
II
Kinesin-like, expressed solely in neurons
Spindle positioning, chromosome alignment and
T09A5.10
II
segregation
Kinesin, role in chromosome segregation and
W02B12.7
II
germline development
Y54G11A.8
II
Putative kinesin light chain
Predicted monomeric kinesin and member of the
R144.1
III
UNC-104 family
Related to XKCM1/MCAK, required to pull
K11D9.1
III
mitotic spindles asymmetrically at anaphase
C45G3.1
I
40
Gene
Sequence
Chr.
Brief Description26
Cytoskeleton-related (cont.)
osm-3 (klp-2; caf-1)
M02B7.3
IV
zen-4 (klp-9)
M03D4.1
IV
klp-18
C06G3.2
IV
klp-10
C33H5.4
IV
vab-8 (klp-5; unc-107)
K12F2.2
V
lfi-1
ZC8.4
X
klp-13
F22F4.3
X
osm-1
T27B1.1
X
Kinesin, required for intraflagellar transport
Kinesin-like, required for polar body extrusion
after meiotic divisions, for completion of
cytokinesis after mitosis, and for formation
and/or maintenance of spindle midzone MTs
Predicted Kinesin similar to human klp2,
required for assembly of meiotic spindles
Predicted Kinesin similar to human klp2,
highly similar to klp-18
Required for many posteriorly-directed cell
migrations, as well as axonal outgrowth and
pathfinding
Interacts with LIN-5, which is essential for
proper spindle positioning and chromosome
segregation
Atypical kinesin-like,similar to Kip3, which
has been implicated in nuclear migration
Presumptive cargo molecule for kinesin-II,
required for proper assembly of sensory cilia
MT Regulators
bicd-1
figl-1
C43G2.2
F32D1.1
IV
V
cdk-2
W03G9.5
C09D4.3
K03E5.3
W03G9.5
C09D4.3
I
I
I
vps-34 (let-512)
B0025.1
I
ZC581.9
prpf-4 (prp-4)
Y106G6A.1
Y106G6E.1
wts-1 (tag-181)
C34B2.3
cdk-9
ZC581.9
F22D6.5
Y106G6A.1
Y106G6E.1
T20F10.1
C34B2.3
H25P06.2a
I
I
I
I
I
I
I
src-2 (kin-22)
F49B2.5
I
ZK622.1
gcy-12
ZK622.1
F08B1.2
II
II
pink-1
EEED8.9
II
ZK177.2
zyg-1
C25H3.1
T19D12.5
ZK177.2
F59E12.2
C25H3.1
T19D12.5
II
II
II
II
ire-1
C41C4.4
II
sphk-1
C34C6.5
II
Dynein regulator
Required for persistence of the germline
Kinases
Protein kinase PCTAIRE and related kinases
Casein (serine/threonine/tyrosine) kinase
Casein (serine/threonine/tyrosine) kinase
Required for vesicular trafficking:endocytosis,
apoptotic cell clearance, and autophagy
Regulation of axon navigation
U4/U6-associated splicing factor
MEKK and related serine/threonine kinases
Glycogen synthase kinase-3
WarTS/lats-like serine threonine kinases
Casein (serine/threonine/tyrosine) kinase
Thought to form transcription factor P-TEFb
SRC oncogene related, non-receptor protein
tyrosine kinase
Protein tyrosine kinase
Membrane guanylyl cyclase
PINK homolog implicated in Parkinson’s,
required for stress response, mitochondrial
homeostasis, CAN neurite growth, and brood
size
Serine/threonine kinase (haspin family)
Required for daughter centriole formation
No description available
No description available
Required for the unfolded protein response
(UPR) in response to ER stress
SPHingosine Kinase
41
Gene
Sequence
Chr.
Kinases (cont.)
M176.9
M176.9
II
gcy-1
AH6.1
II
wee-1.1
F35H8.7
II
wee-1.3
Y53C12A.1
II
old-2 (tkr-2; kin-28)
ZK938.5
II
R05H5.4
C14A4.13
Y39G8C.2
R05H5.4
C14A4.13
Y39G8C.2
II
II
II
pat-4
C29F9.7
III
kin-19
C03C10.1
III
col-94
K11H12.9
F52C12.2
R13H9.5
R13H9.6
C39H7.1
kin-26
W05B2.1
K11H12.9
F52C12.2
R13H9.5
R13H9.6
C39H7.1
T06C10.6
III
IV
IV
IV
IV
IV
IV
gcy-8
C49H3.1
IV
pmk-2
pmk-3
pct-1 (pct)
pkg-2
C49C8.1
F08B4.3
F49C12.7
cka-1
kin-24
F44D12.11
F42G8.3
F42G8.4
C07G1.3
C09G4.2
C49C8.1
F08B4.3
F49C12.7
C28D4.2
K07F5.4
F44D12.11
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
frk-1
T04B2.2
IV
C04G2.2
Y43C5B.2
T25B9.5
ZK593.9
C27D8.1
C04G2.2
Y43C5B.2
T25B9.5
ZK593.9
C27D8.1
IV
IV
IV
IV
IV
pik-1
K09B11.1
IV
C49C3.2
gcy-27
bbs-8
C49C3.2
C06A12.4
T25F10.5
IV
IV
V
42
Brief Description26
No description available
Receptor-type, transmembrane guanylyl
cyclase needed for chemotaxis
WEE homolog which regulates cell size by
inhibiting entry into mitosis
WEE homolog which regulates cell size by
inhibiting entry into mitosis
transmembrane protein tyrosine kinase, affects
mean and maximum life span
No description available
No description available
No description available
Required for formation of integrin-mediated
muscle cell attachments during embryogenesis
Wnt signaling pathway, regulates endoderm
formation and spindle orientation
COLlagen. No other description available
No description available
No description available
No description available
No description available
No description available
No description available
Regulates thermotaxis via the AFD
thermosensory neurons
p38 MAPK homolog, stress response
p38 MAPK homolog, osmotic stress activated
PCTAIRE class cell cycle kinase.
Protein Kinase, cGMP-dependent
No description available
No description available
No description available
Choline kinase
No description available
No description available
Required early for embryonic cell
proliferation and later in embryonic epidermal
cells for enclosure, morphogenesis and late
stages of differentiation
No description available
No description available
No description available
No description available
No description available
pelle/IRAK kinase homolog that may be
involved in apoptosis
No description available
No description available
Required for cilia biogenesis and function
Gene
Sequence
Chr.
Kinases (cont.)
gcy-20
Y59A8A.b
gcy-22
T08D2.7
F56A4.5
F21H7.9
Y59A8A.b
T03D8.5
T08D2.7
F56A4.g
V
V
V
V
V
mrck-1
K08B12.5
V
scd-2
T10H9.2
V
F09G2.1
F09G2.1
V
air-1
K07C11.2
V
F38E1.3
E02C12.6
E02C12.7
E02C12.10
C50F4.1
C50F4.10
C50H2.7
F38E1.3
E02C12.6
E02C12.7
E02C12.10
C50F4.1
C50F4.10
C50H2.7
V
V
V
V
V
V
V
gcy-6
B0024.6
V
F32D8.10
F58B4.5
pak-2
F32D8.10
F58B4.5
C45B11.1
V
V
V
daf-11
B0240.3
V
rskd-1
R90.1
R10D12.10
T08G5.2
tag-191
F55C5.7
R90.1
R10D12.10
T08G5.2
C53A5.4
V
V
V
V
V
T01G5.1
T01G5.1
V
F28C10.3
pifk-1
F47F2.1
F28C10.3
F35H12.4
F47F2.1
X
X
X
lin-18
C16B8.1
X
hpk-1
F20B6.8
X
cka-2
dgk-2
C52B9.1
F46H6.2
X
X
Brief Description26
Predicted guanylate cyclase
No description available
Predicted guanylate cyclase
No description available
No description available
MRCK and DMPK ortholog, regulates
embryonic elongation through myosin
regulatory light chain during development
Regulate dauer formation via activation of the
daf-3 transcription factor or TGF-β pathway
No description available
Aurora-A family, required for embryonic
survival, germline proliferation and
development, locomotion, and vulval
development; implicated in centrosome
separation and spindle assembly
No description available
No description available
No description available
No description available
No description available
No description available
No description available
gcy-6 encodes a predicted guanylate cyclase;
expressed in the ASEL neurons
No description available
No description available
Putative p21-activated kinase
Required for chemosensory function
dependent processes, including dauer
formation and recovery and chemotaxis, and
axon formation
Ribosomal protein S6 Kinase Delta homolog
No description available
No description available
No description available
No description available
KIN-16 family of receptor protein tyrosine
kinases
No description available
PhosphoInositide Four Kinase (PI-4 Kinase)
No description available
Required for establishing the polarity of the
secondary vulval cell lineage, may be a
receptor for Wnt-like signaling molecules
Predicted dual-specificity protein kinase,
distant homolog of DYRK1A
Choline kinase
Putative diacylglycerol kinase.
43
Gene
Sequence
Brief Description26
Chr.
Kinases (cont.)
svh-2
C36B7.1
C55B6.5
T14E8.1
C36B7.1
C55B6.5
X
X
X
sek-1
R03G5.2
X
kin-9
VZC374L.1
pkn-1
F59F5.3
F08F1.1
VZC374L.1
F46F6.2
F59F5.3
X
X
X
X
abl-1
M79.1
X
C44C10.7
C44C10.7
X
mes-1
F54F7.5
X
T24D5.4
C29F7.2
T24D5.4
C29F7.2
X
X
sad-1
F15A2.6
X
gck-4
F16B12.5
K04C1.5
E02H4.3
C04A11.3
F16B12.5
K04C1.5
E02H4.3
X
X
X
X
mbk-1
T04C10.1
X
piki-1
F39B1.1
X
kin-20
F22H10.1
F22H10.5
F46F2.2
F22H10.1
F22H10.5
X
X
X
aak-2
T01C8.1
X
Suppressor of VHp-1 deletion lethality
No description available
No description available
MAPKK, activates both JNK-1 and PMK-1 in the yeast
Hog pathway
Predicted protein tyrosine kinase
No description available
Protein Kinase N (PKN) homolog
No description available
Inhibits germline apoptosis induced by radiation or by
natural aging; required for germline apoptosis induced
by oxidative, osmotic or heat-shock stress, and for
pathogenesis by S. flexneri infecting the intestine
No description available
Required for unequal cell divisions in the early
embryonic germline; involved in positioning of the early
mitotic spindle and of associated P granules
No description available
No description available
Required for several aspects of presynaptic development,
functions in a complex with STRD-1, to regulate
axonal-dendritic polarity and synapse organization
Germinal Center Kinase family
No description available
No description available
No description available
Predicted dual-specificity protein kinase, DYRK1A
ortholog
Redundantly regulates apoptotic cell clearance
presumably through regulation of PtdIns(3)P
No description available
No description available
No description available
Positively regulates adult lifespan, negatively regulates
germline proliferation during dauer development
Synaptonemal-Complex-Related
syp-2
C24G6.1
V
Structural component of the SC; required for maintaining
stable pairing between homologous chromosomes during
meiosis
44
Table 3 List of positive hits with emb phenotype
Gene
Sequence
Chromatin-related
smo-1
taf-4
let-526 (C01G8.8)
hmg-3
ekl-1
dcp-66
K12C11.2
R119.6
C01G8.8
C32F10.5
F22D6.6
C26C6.5
Chr.
I
I
I
I
I
I
HTP-3 IP
him-1 (smc-1)
egrh-1
F28B3.7
C27C12.2
I
X
HIM-3 IP
aspm-1 (tag-255)
cap-1 (capz/a)
C45G3.1
D2024.6
Cytoskeleton-related
kca-1
C10H11.10
lin-5
T09A5.10
klp-7
K11D9.1
zen-4 (klp-9)
M03D4.1
klp-10
C33H5.4
MT Regulators
figl-1
F32D1.1
I
IV
I
II
III
IV
IV
V
Kinases
cdk-2
C09D4.3
ZC581.9
cdk-9
zyg-1
wee-1.3
mrck-1
K03E5.3
C09D4.3
ZC581.9
H25P06.2a
F59E12.2
Y53C12A.1
K08B12.5
I
I
I
I
II
II
V
Synaptonemal-Complex-Related
syp-2
C24G6.1
V
Table 4 List of positive hits with him phenotype
Gene
him-1 (smc-1)
Sequence
HTP-3 IP
F28B3.7
Chr.
I
45
Table 5 List of negative hits with emb phenotype
Gene
Sequence
Chromatin-related
pop-1
B0261.1
Chr.
W10C8.2
B0261.1
I
I
HTP-3 IP
spd-5
cdc-37
cyk-1
F56A3.4
W08F4.8
F11H8.4
I
II
III
HIM-3 IP
met-2
rsa-2
R05D3.11
Y48A6B.11
Cytoskeleton-related
klp-15
M01E11.6
klp-16
C41G7.2
klp-19
Y43F4B.6
epi-1 (egf-3; lgx-3)
K08C7.3
arx-2 (arp-2)
K07C5.1
MT Regulators
mei-1
T01G9.5
spd-2
F32H2.3
zyg-9
F22B5.7
tac-1 (2P40)
Y54E2A.3
cls-2
R107.6
tbg-1 (tubg; sas-3)
F58A4.8
III
III
I
I
III
IV
V
I
I
II
II
III
III
Kinases
dapk-1 (mor-3; tag-119)
let-502
ppk-1
bub-1
ZC581.2
ZC581.7
T21G5.1
smg-1 (mab-1)
gska-3
dyf-5
csnk-1
pkc-3
F59E12.3
C03C10.2
B0218.5
W01B6.2
chk-1
K12C11.4
C10H11.9
F55A12.3
R06C7.8
ZC581.2
ZC581.7
T21G5.1
C48B6.6
C36B1.10
M04C9.5
Y106G6E.6
F09E5.1
F59E12.3
C03C10.2
B0218.5
W01B6.2
Y39H10A_224.a
I
I
I
I
I
I
I
I
I
I
I
II
II
III
IV
IV
V
46
Table 6 List of positive hits with roles in MT motor activity
Gene
Sequence
Cytoskeleton-related
dhc-1
klp-17
klp-6
klp-7
osm-3 (klp-2; caf-1)
zen-4 (klp-9)
klp-18
klp-10
vab-8 (klp-5; unc-107)
klp-13
T21E12.4
W02B12.7
R144.1
K11D9.1
M02B7.3
M03D4.1
C06G3.2
C33H5.4
K12F2.2
F22F4.3
Chr.
I
II
III
III
IV
IV
IV
IV
V
X
47
Table 7 List of genes untested due to unavailability
Gene
ZC247.1
lec-1
nurf-1
smc-3
pix-1 (tag-207)
Sequence
HTP-3 IP
Chr.
ZC247.1
W09H1.6
F26H11.2
Y47D3A.26
K11E4.4
I
II
II
III
X
Gene
F59A6.4
T05A7.6
T05A7.6
Y51B9A.9
N/A
N/A
N/A
kin-16
R09D1.13
N/A
Y47D3A.r
Y75B8A.a
Y69F12A_449.c
lit-1
spk-1
sel-5
C15H7.2
N/A
N/A
egl-4
unc-43
unc-22
ZK354.6
ZK596.2
ZK666.8
pmk-1
jnk-1
N/A
ark-1
N/A
N/A
N/A
dgk-4
N/A
akt-1
dkf-2
unc-60
Y116F11.zz34
Y60A3.b
K09C6.1
K11C4.1
Y42A5A.4
kin-33
N/A
HIM-3 IP
ifb-2
mat-1
ZK1067.2
F10C1.7
Y110A7A.17
ZK1067.2
II
II
II
Cytoskeleton-related
nud-2
spd-1
tpxl-1
arp-1
gpr-1 (ags-3.2)
klp-20
zyg-8 (apo-1)
klc-1
klc-2
ebp-1
ebp-3 (tag-201)
R11A5.2
Y34D9A.4
Y39G10AR.12
Y53F4B.22
F22B7.13
Y50D7A.6
Y79H2A.11
M7.2
C18C4.10
Y59A8B.7
Y59A8B.9
I
I
I
II
III
III
III
IV
V
V
V
Kinases
kin-1
dkf-1
R06A10.4
ZK524.4
C24G7.5C
rskn-2
rskn-1
F33D11.7
ZK507.2+
H05L14.1
Y18D10A.f
air-2
kin-33
N/A
N/A
C35E7.10
kin-23
kin-23
H05L14.1
let-363
Y39G8_112945
F59A6.4
ZK909.2a
W09C5.5
R06A10.4
ZK524.4
C24G7.5C
C54G4.1C
T01H8.1aC
F33D11.7
ZK507.2+
H05L14.1C
Y18D10A.f
B0207.4
B0205.7
Y71F9B_297.b
Y39G10A_244.a
C35E7.10a
W04G5.6C
W04G5.6N
H05L14.1N
B0261.2
Y39G8_112945
F59A6.4C
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
48
Sequence
Kinases (cont.)
F59A6.4N
T05A7.6C
T05A7.6N
Y51B9A.9
Y38E10_05694
Y38F1A.l
Y62F5_01369
M176.7
R09D1.13
Y53F4B.b
Y47D3A.r
Y75B8A.a
Y69F12A_449.c
W06F12.1a
B0464.5
F35G12.3a
C15H7.2
Y116A8_02435.1
Y55D5A_392.a
F55A8.2a
K11E8.1a
ZK617.1a
ZK354.6
ZK596.2
ZK666.8
B0218.3
B0478.1
Y105C5.y
C01C7.1
C30F8
W02A2.d
Y69E1A.c
F42A9.1a
Y105C5_11465
C12D8.10a
T25E12.4a
C38C3.5B
Y116F11.zz34
Y60A3.b
K09C6.1
K11C4.1
Y42A5A.4
DC2.7C
DC2.7N
Chr.
II
II
II
II
II
II
II
II
II
II
III
III
III
III
III
III
III
III
III
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
V
V
V
V
V
V
V
V
V
V
Gene
R02C2.1
R02C2.1
N/A
N/A
N/A
T08G5.2
N/A
C24G6.2
nipi-4
T10B5.2
E02C12.12
H37A05.2
gcy-14
akt-2
pdk-1
pkc-2
sgk-1
lin-2
ZC373.4
F16B12.7
ZC373.3
F39F10.2
F39F10.3
cdk-4
sma-5
C36B7.1
jkk-1
sid-3
T24D5.4
W02H3.2
C29F7.1
dgk-1
gcy-9
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Sequence
Kinases (cont.)
R02C2.1C
R02C2.1N
Y59A8_01348+
Y58A7A.f
Y60A3.y
T08G5.2
Y50D4B_4.a
C24G6.2a
F40A3.5
T10B5.2
E02C12.12
H37A05.2
ZC412.2
F28H6.1
H42K12.1
E01H11.1
W10G6.2
F17E5.1a
ZC373.4
F16B12.7
ZC373.3
F39F10.2
F39F10.3
F18H3.5a
W06B3.2
C36B7.1
F35C8.3
B0302.1
T24D5.4
W02H3.2
C29F7.1
C09E10.2a
ZK455.2
Y50D7.Con462
Y39G10.con155
Y32H12A_68.a
Y105E8
Y113B8_04829
F58D5
Y116A8_02435.2
AC8.b
D2024.su1
D2024.su2
Y40A1A.17
DC2.su1
Y45G12C.k
Gene
Chr.
N/A
N/A
Dead Gene
Dead Gene
V
V
V
V
V
V
V
V
V
V
V
V
V
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
49
Sequence
Kinases (cont.)
Y75B8A.aa
Y67D8.Contig183
F40G9.13
Y37H2A.a
Chr.
N/A
N/A
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