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MODIFICATION OF THE SPD-2 PROTEIN AND ITS
ROLE DURING CENTROSOME ELIMINATION IN
C.elegans
The Roy laboratory – Developmental control of the cell cycle
Mc GILL UNIVERSITY - MONTREAL
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MODIFICATION OF THE SPD-2 PROTEIN AND ITS ROLE DURING CENTROSOME
ELIMINATION IN C.elegans
Sexually reproducing organisms must eliminate a pair of centrosomes prior to the first zygotic
division to avoid the formation of a multipolar spindle. During the development of the C.
elegans germ line, germ cells undergo several developmental changes following oocyte
specification that include the elimination of the centrioles that were present during the earlier
meiotic stages. This process requires the activity of a p21/p27-like CDK inhibitor protein
called cki-2. Moreover, a cyclin E/CDK2-dependent phosphorylation is required to stabilize
the centrioles and its cki-2-dependent inhibition results in their rapid
destabilization/elimination. Many of the proteins required for centrosome duplication possess
canonical CDK phosphorylation sites and could be candidate proteins for a potential role as
CDK targets for centriole stabilization. Among them, SPD-2, acting upstream the centriole
assembly pathway is able to shift from perinuclear foci to nuclei during oogenesis, suggesting
that it is modified in such a way that influences its localization.
INTRODUCTION

Centrosome and centrioles
Sans titre1In animal cells, the function of microtubule-organizing centers (MTOCs) is mainly
assumed by centrosome (1). This organelle, discovered at the turn of the twentieth century and first
studied by Theodor Boveri (2) comprises, as illustrated in Figure 1, two distinct structures: a pair of
centrioles, the main core constituents of the centrosome, surrounded by a filamentous network of
proteins, called pericentriolar material (PCM). Centrioles play a crucial role in PCM assembly and
consequently, in determining the MTOC number (1). Molecular epistasis experiments, which have
Figure 1. A: C. elegans centrosome structure. B: Sequential recruitment of proteins during C. elegans
centriole assembly: SPD-2, which localizes both to PCM (PeriCentriolar Material) and centrioles, recruits
ZYG-1 kinase to the centriole Together, they make it possible the incorporation of structural components of the
central tube: SAS-5 and SAS-6. Finally, SAS-4 supports the tethering of microtubules to the central tube,
allowing PCM recruitment and organization (3).
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been successfully performed in C. elegans for a few years, showed that their assembly required the
sequential action of four coiled-coil proteins and one kinase: SAS-4, -5, -6, SPD-2 (SPindle Defect)
and ZYG-1 (ZYGote defective embryonic lethal), respectively (Figure 1).
Even though centrioles are different in nematodes compared to Vertebrates, because of their
short size, their singlets of microtubules (instead of doublets) and their lack of –Tubulin and
Centrin (two major centrosome proteins in Vertebrates), C. elegans has emerged in recent years as
an attractive model system for studying centrosome duplication. Using genetic analyses of events
that occur in the one-cell embryo in C. elegans, researchers were able to identify essential
components for centriole formation; in some of which, including SAS-4, SAS-6 and SPD-2, share
homologies with centriolar proteins of mammals. The morphological properties of the one-cell
stage embryo and a clear size difference between centrioles and the PCM allow analysis of
centrosome duplication in living specimens with high resolution as well as studies of gene function
of centrosome components (by fluorescence imaging or by RNA interference), respectively (4).

Centrioles duplication and cell cycle
Centrioles from both nematodes and other metazoans play crucial roles in cellular
mechanisms. Their main function is to provide a strong solid core around which more amorphous
PCM can be structured, a template
structure needed to produce a new
centriole, or an anchor for microtubules
in the PCM, which communicate force
from the cortex to the nucleus and then
along the spindle guaranteeing that
equivalent forces will be exerted from all
sides onto the attachment point of these
microtubules
during
cytokinesis.
Moreover, centrioles act as a cell cycle
regulator consistent with their role as a
Figure 2. Centriole and centrosome duplication
during cell cycle. In red: checkpoints of cell cycle
; in green, blue and pink: other controls of
centrosome duplication; in black: phases of the cell
cycle
scaffold onto which signaling molecules
may be concentrated and coordinated (5).
Such functions suggest that their number
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must be strictly regulated and intimately linked to the cell cycle (6). While the mechanism of their
duplication is still only partially understood (4), it was shown that different levels of controls,
involving some kinases, occur throughout the cell cycle. Firstly, the centriole-centrosome
duplication cycle seems to be regulated by canonical checkpoints of the cell cycle, as illustrated by
the red stars on Figure 2. Secondly, two rules could be determined: on the one hand, centrosomes
duplicate once and only once per cell cycle, in synchrony or not with DNA during the S phase (1,6).
This property is ensured by Separase (Figure 2, in green), a protease that prevents a new round of
duplication from S phase to mitosis. On the other hand, in order to avoid the production of
excessive numbers of centrioles, a phenotype commonly observed in cancer cells, there is only one
progeny centriole next to each parental centriole, thanks to the combined actions of the ZYG-1
kinase and the Cdk-2/cyclin A and/or E complex (Figure 2, in pink and blue). Finally, some
phosphatases are expected to counteract these kinase actions. (6)
In addition, centrosome biogenesis can follow a route different from the canonical centrosome
duplication cycle, as described in Figure 2. This is the case in cancerous cells in Mammals and
during normal development in other metazoans: such as endocycling cells in Drosophila or the
intestinal cells and spermatocytes in C. elegans. In animals, fertilization requires the fusion of two
haploid gametes resulting in a zygote containing only one full centrosome, so that its first mitosis
leads to the formation of two centrosomes. This result can be obtained by different strategies
depending on the species. In C. elegans, the centrioles disappear during oogenesis and
spermatocytes lose their PCM (4). As in other species, the mechanism for centriole disappearance is
unknown. However, it was recently shown by genetic experiments that a p21/p27-like cyclindependent kinase (Cdk) inhibitor (Cki-2) was needed for maternal centriole elimination during
oogenesis. Indeed, loss of cki-2 results on the one hand in misregulation of cyclin E/Cdk-2 activity,
and on the other hand in the inheritance of centrosome during oogenesis, suggesting that targets of
Cdk activity are involved in centriolar perdurance (7).

Spd-2
It was previously shown that in C. elegans deficient for cki-2 by cosuppression reverse genetic
experiments (cki-2cs), SPD-2 persisted throughout oogenesis (7). Bioinformatic analysis of proteins
needed for centriole assembly demonstrated that all the proteins (SAS-4, SAS-5, SAS-6, ZYG-1 and
SPD-2) involved in centriole assembly contained a pattern of Cdk-phosphorylation sites (annexe,
Figure 8). Therefore, might the SPD-2 coiled—coil protein (known to act upstream of the centriole
assembly pathway) be a substrate for cyclin E/Cdk-2 and by this way act upstream of centriolar
disassembly? Recently, a study describing SPD-2 functional domains has briefly shown that
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varying amounts of SPD-2 could be present in the nuclei of oocytes (8, Figure 3A) whereas
centrioles have already disappeared from the cytoplasm and whereas SPD-2 clusters generally
appear as perinuclear foci in dividing cells (8). We can thus wonder if SPD-2 is modified during
the cell cycle and what its role is if it is involved in centrosome elimination.
To answer these questions, my first work was to confirm a change in SPD-2 localization from
the cytoplasm to the nucleus by immunofluorescence, and, if confirmed, at what step of oogenesis
the shift occurs. Then, I attempted to discover what modification allowed this move. Using
Western Blots, I tried to identify different forms of denatured SPD-2 protein but I have not
managed to demonstrate it yet because of some experimental constraints. Then, I envisaged to
express a genomic spd-2 sequence comprising a mutated phosphorylation site using bombardment,
to start a new immunofluorescence assay and to perform a new Western Blot using SPD-2
antibodies in order to determine if the shift observed was really due to a phosphorylation event.
However, this experiment is still underway.
RESULTS

Spd-2 shifts from the cytoplasm to the nucleus during oogenesis
My objective was to look for the presence of SPD-2 coiled-coil protein in the nucleus of
oocytes (8). I performed an immunofluorescence labelling of SPD-2::GFP in dissected and fixed
gonads from adult spd-2::spd-2::GFP worms using monoclonal antibodies against GFP (Figure 4).
By microscopy, I could identify SPD-2::GFP in the germline but also, later, in the pachytene, where
centrosome are absent (Figure 3). Moreover, It was shown that SPD-2::GFP foci shifted from a
perinuclear region in the hermaphrodite mitotic germ cells (Figure 4, A) to the nuclei of late
pachytene cells during oogenesis (Figure 4, B).
Figure 3. Adult hermaphrodite germline of C. elegans. Extruded gonads stained with DAPI. At adult
stage, wild-type hermaphrodite C.elegans worms produce both female and male gametes. The female germ
line is divided into: a "distal mitotic" region made of dividing germ cells, a "transition zone" where germ
cells enter meiosis and form a germline syncitium, a "pachytene zone" where the meiotic syncitium grows
and where DNA begins condensing. Finally, in proximal zone, cells undergo diakinesis to become oocytes,
i.e cells arrested in meiotic prophase I where six chromosomes are clearly visible (9).
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Figure 4. SPD-2-GFP persists in the later stages of pachytene and shifts from the
perinuclei to nuclei during oogenesis. (A-D) extruded gonads from a strain expressing
GFP-SPD-2 and stained for GFP (green-sometimes appears yellow due to overlap with red
on merged picture). SPD-2 concentrates in perinuclear foci (arrows) in the distal portion of
the hermaphrodite germline (A). Such foci are absent in oocytes (B) and shift into nuclei, in
late pachytene (B-D).
In parallel, an immunofluorescence labeling of SPD-2 in dissected and fixed intestines from
different stages of N2 worms was performed with polyclonal antibodies against SPD-2 (Figure 5).
By microscopy, it was shown that SPD-2 perinuclear foci, present in diploid (2n) intestinal cells
from the L1 stage, shifted into the nuclei in polyploid (8-16n) intestine cells from L2-L3 stage
(Figure 5, B), where centrioles are absent (annexe, Figure 9).
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Int 3-7
Figure 5. SPD-2 localization changes from centriolar to nuclear foci at the end of the L2
stage in intestinal nuclei. (A) Post-embryonic cell cycle of intestinal nuclei. The intestinal
cells do not undergo proliferative postembryonic cell division. During the L1 molt, some
intestine cells (Int 3-7) undergo DNA replication then, perform karyokinesis without
cytokinesis. After the karyokinesis, all the nuclei undergo one round of endoreplication.
Endoreplication occurs at each of the following larvae molts, so that each of the intestinal
nuclei in the adult is 32C (9) . (B) Extruded intestine from N2 larvae stained with polyclonal
antibodies against SPD-2. SPD-2 concentrates in perinuclear foci (arrows) in L1 intestine
cells. Such foci are absent in L2-L3 while diffuse SPD-2 is visible in intestinal nuclei
(unpublished Yu Lu's data).
This experiment, done both in the germline and in intestinal cells, confirms the ability of
SPD-2 to shift into the nuclei from the perinuclear foci during centriole elimination in C. elegans.

SPD-2 coiled-coil protein contains CDK- targeted phosphorylation sites
Since SPD-2 acts upstream of centriole assembly and centriole disappearance during
oogenesis seems to be linked to the inhibition of Cyclin-dependent kinase 2 by CKI-2 (7), the
nuclear form of SPD-2, observed by immunofluorescence, might result from a modification made
by Cdk2, meaning that it might be phosphorylated. According to a previous study (11) the presence
of six conserved Cdk phosphorylation sites (both in C. elegans and C. briggsae) was expected in
the nematode SPD-2 sequence. I confirmed, in silico their presence (aa positions: 143, 171, 220,
233, 259 and 545) in C.elegans SPD-2, using a new Group-based Phosphorylation (GPS) Scoring
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method (12). Moreover, by this way, I could classify the most probable Cdk-phosphorylation sites
according to their GPS score (Figure 6).
Among the twelve phosphorylation sites found, only one, located on the Threonine 233 had a
GPS score higher than 8 (Figure 6). Thus, Thr 233 is the most probable Cdk-phosphorylation site
on SPD-2, if considering that conservation of sequences throughout evolution is evidence of
function.
Figure 6. The most probable Cdk-phosphorylation sites of SPD-2 sequence, found by the
Group-based Phosphorylation Site (GPS) scoring method (12). GPS scoring is a statistical
method. Best scores correspond to the most probable phosphorylation sites. Threonine 233 is
the only amino acid with a GPS score higher than 8. Small stars surmount amino acids most
likely to be targeted by CDKs. Only scores higher than 4 were considered as significant.

Different forms of SPD-2 protein might coexist during C. elegans development
Having previously demonstrated that SPD-2 contains Cdk-phosphorylation sites and that the
protein shifts from the cytoplasm to the nuclei in some C. elegans tissues (germ line and intestine),
it can be speculated that the passage of SPD-2 into different cellular compartments (cytoplasm and
nuclei) during post-embryonic development might be due to a modification of the protein, such as a
phosphorylation.
In order to determine the presence of any modified SPD-2, I first looked for the presence of
different forms of SPD-2 in wild-type worms, by Western Blot, using a polyclonal antibody against
SPD-2 (8). In order to know if SPD-2 undergoes modifications specific to the development of the
germ line or intestine, I isolated proteins from different larval stages in wild-type C. elegans
hermaphrodites. More exactly, I compared SPD-2 from the L1 stage, where there are only mitotic
germ cells (dividing cells where centrioles are present, so where SPD-2 is located only in
perinuclear foci), to SPD-2 taken from young adults where gonads are completely functional and in
which I had observed the presence of SPD-2 from late pachytene nuclei. Moreover, I performed the
same experiments with worms in the L3/L4 transition stage, when the oocytes are not yet present,
but where pachytene nuclei are well established, unlike the L1 stage. In this way, I could
determine wether the quantity of modified SPD-2 is linked to oogenesis (Figure 7).
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spd-2
(RNAi)
Figure 7. Western Blot analysis of protein extracts of wild-type C. elegans. Total protein
extracts were prepared from L1, L3/L4 and young adult N2 worms, and spd-2(RNAi)
animals. 5l (lane 3) or 15l (lane 1 and 2) of protein extract was separated by SDS-PAGE
and the presence of SPD-2 and -Tubulin (positive control) was measured by Western blot
(primary antibodies: polyclonal anti-SPD-2 (8), anti--Tubulin; secondary antibodies: antirabbit and anti-mouse, respectively)
The molecular weight of SPD-2 has been estimated at 91.5kDa (13). However, the bands that
observed on the Western Blot indicate 60kDa and 85kDa proteins, regardless of the age considered
(Figure 7, lanes 1-3). Moreover, an additional band located at 60-85kDa is visible in young adult
worms (Figure 7, lanes 3). However, it is impossible to know what bands correspond to SPD-2
because none of them indicate SPD-2 molecular weight (91.5kDa). To solve this question, I
compared protein extracts from N2 worms to those from adult hermaphrodites worms fed with E.
coli expressing spd-2 dsRNA. Using spd-2 (RNAi), I could determine, by observing any missing
bands, which one(s) correspond(s) to SPD-2. However, my results were not convincing (Figure 7,
lane 4). Indeed, I do not observe any differences between protein extracts from spd-2 (RNAi)
animals and those from wild-type worms. Therefore, I have not been able to conclude anything yet.

Modification of Cdk-targeted phosphorylation site in SPD-2 sequence
Another way to determine wether Cdk-phosphorylation affects SPD-2 activity in centriole
disassembly is to modify Threonine 233, the most probable Cdk-pphosphorylation site on SPD-2,
by an amino acid that cannot be phosphorylated. The substitution of Threonine 233 by a Valine
(Threonine and Valine differ only by the presence of a kinase-targeted hydroxyl group on
Threonine lateral chain) was performed by high throughput site-directed mutagenesis. A PCR
performed with a mutagenic oligonucleotide primer generated a mutated codon (ACT into GTA,
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according the genetic code) in the spd-2 genomic sequence from pMR811. Then, the plasmid was
bombarded into living unc-119 (ed3) mutant worms (viable and uncoordinated) to allow the
integration of a few copies of the mutated spd-2 transgene into the genome. Only unc-119 (ed3)
animals transformed with the plasmid containing the wild-type unc-119 gene survive starvation (on
the contrary, unc-119(ed3) cannot survive dauer formation) while non-transformed animals and
those that had lost unstable arrays do not survive .Then, modifications on the mutated SPD-2::GFP
and any location changes during centriole elimination can be analyzed as previously described
(fluorescence imaging and Western Blot). However, this experiment is still underway.
DISCUSSION & PERSPECTIVES
1. DISCUSSION
What mechanism governs the centriole elimination during oogenesis? It was previously
demonstrated that SPD-2 acts upstream of centriole assembly (3, 18) but when centrioles disappear
during oogenesis, SPD-2 is not eliminated at the same time (7, 8). Therefore, we can wonder why it
is not immediately degraded and what happens. In this study, I have demonstrated that SPD-2::GFP
protein shifts from the cytoplasm to the pachytene nuclei during centriole elimination in wild-type
hermaphrodite C. elegans. In order to show the change of behavior of SPD-2, I used the
fluorescence imaging but I had to overcome some technical difficulties. Firstly, I tried to detect
SPD-2-GFP either in living adult pie-1::GFP::spd-2 worms or in dissected and fixed gonads (DAPI
staining), using a compound microscope. However, the signal was too weak and there was a strong
background, probably because of the small size of centrosome and of the autofluorescence that is
usually observed in living worms. Then, I performed immunofluorescence to label SPD-2 in N2.
But I could not obtain satisfying pictures since background fluorescence level was too elevated.
Only staining SPD-2::GFP transgenic animals with monoclonal antibodies against GFP led to
convincing results. Yet, it was previously described that an immunofluorescence assay in female
germ line with the same antibodies against SPD-2 than those used in this study, did not give as
much noise, if it was analyzed by confocal microscopy (8). A confocal microscope consists,
basically, of a high quality compound microscope with a laser illuminator, electronic image detector
and computer for image storage and processing, with an adjustable confocal pin hole in the imaging
plane. Hence, confocal microscope allows a high-resolution epi-fluorescence microscopy, the
acquisition of thin optical sections of finite, but controlled thickness and the decrease of the
background fluorescence level of the samples. Thus, a more precise analysis with confocal
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microscope may confirm the presence of SPD-2 in the late pachytene of N2 germ lines, using
antibodies against SPD-2 instead.
Is SPD-2 modified during oogenesis in wild-type worms? Is the location change of SPD-2
during centriole elimination linked to physical modifications of the protein? In order to discover if
SPD-2 could be modified during oogenesis, I performed Western Blots to identify the presence
different forms of denatured SPD-2 protein at different stages of C. elegans development with
antibodies against SPD-2. I evidenced several bands that conflict with the predicted size of SPD-2.
Therefore, I then performed a Western blot comparison between proteins from spd-2(RNAi) worms
(10) with proteins from N2. However, contrary to what I expected, no band disappeared in spd-2
(RNAi) animals (while spd-2(RNAi) should have prevented the traduction of SPD-2 and the missing
band should have indicated what band corresponds to SPD-2). Having used spd-2 (RNAi) by
feeding and knowing this method produces slightly more variable results than RNAi by soaking or
injection (9), I am currently performing again western blot with new spd-2 (RNAi) worms. Other
troubleshooting can reduced the observed background: on one hand, apply the antibody to the
sample once or twice may reduce the unspecific binding; on the other hand, since it is possible that
repetitive uses of protein samples generate proteolysis, it is necessary to use fresh protein extract.
Finally, no firm conclusion on modifications of SPD-2 during post-embryonic development can be
drawn from my analysis.
However, it was demonstrated that there are Cdk-phosphorylation sites within SPD-2 sequence
contains and that the most probable of them are conserved in other nematodes (12). So, if it is
expected that SPD-2 can be regulated by Cdk-phosphorylation, a more precise phosphorylation
analysis of SPD-2 could be performed by Mass spectrometry. A Large-scale identification of C.
elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry performed
by a Japanese team, in 2003 (14) has demonstrated that among the approximately 5,400 peptides
assigned in this study, many peptides with post-translational modifications, such as N-terminal
acetylation and phosphorylation, were detected. Indeed, the phosphorylation of SPD-2 may have
already been characterized and it would be interesting to obtain an English version of their article.
2. PERSPECTIVES
Does Cyclin E/CDK2 target SPD-2 or other centriolar components?
If it is soon demonstrated that SPD-2 can be phosphorylated and that this phosphorylation
corresponds to centriole elimination, it will be then necessary to demonstrate if SPD-2 is a target of
cyclin E/Cdk2. That SPD-2 is a target of Cdk2 suggests a physical interaction between the two
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proteins. In order to confirm this physical interaction, it would be interesting to use reverse
proteomic approaches, like the yeast two-hybrid system (9).
However, if this method shows that the complex cyclin E/Cdk2 is not responsible for the
phosphorylation of SPD-2, it would be interesting to investigate which kinase activity is involved in
SPD-2 phosphorylation. In the C. elegans embryo, loss of spd-2 gene activity results in a failure of
centrosome duplication. As a consequence, bipolar spindles are not assembled; DNA is not
segregated, cytokinesis fails and the embryos die (15). To identify factors that interact with spd-2
to regulate centrosome duplication, a sensitive genetic suppressor screen could be designed to
identify mutations that restore normal centrosome duplication to an embryo deficient in spd-2
activity, as it was previously done to highlight factors which interacts with ZYG-1, an other kinase
involved in centriole assembly (16). Among these suppressor mutations, we could detect gene
encoding for kinases and then, investigate more precisely how these kinases physically interact with
SPD-2, by the yeast two-hybrid system, as well.
To know if cyclin E/Cdk2 target other centriolar components, it would be necessary to do
again the same experiments than for SPD-2. However, another solution could be the highlighting of
Cdk2 protein complex in C. elegans. A Tandem affinity purification (TAP), performed with the
tagged-Cdk2 could bait its targets among C. elegans proteins and these proteins would be then
analyzed by mass spectrometry.
Is SPD-2 modified to regulate its localization?
Another question rises from the observation of SPD-2 shift from cytoplasm to nucleus: Is a
post-transcriptional modification of SPD-2 responsible for this shift and consequently for centriole
disassembly or on the contrary, is this probable modification responsible for centriole disassembly
and indirectly for SPD-2 sending in nucleus? To answer, it would be interesting to lead epistasis
genetic experiments with RNAi against each component of centriole assembly (spd-2, zyg-1, sas-4,
sas-5 and sas-6) in order to determine how centriolar proteins act the ones compared to others
during centriole elimination in the germline, for instance, as it was precedently made to determine
sequential protein recruitment in C. elegans centriole formation (3, 18). Thus, we would be able to
know on the one hand, if SPD-2 acts upstream centriole disassembly; on the other hand, if
localization change of SPD-2 results from a direct modification of the coiled-coil protein or from
the modification of one of these partners, like another component of centriole assembly. That is
why Cdk-phosphorylation analysis is currently performed in every known components of centriole
assembly: ZYG-1, SAS-4, SAS-5 and SAS-6.
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On the other hand, as it was recently demonstrated for ZYG-1 (16), a genetic suppressor
screen could highlight the presence of genes encoding for nuclear envelope components among
SPD-2 partners. That could explain the presence of SPD-2 inside nucleus during oogenesis or
intestinal cells development.
Do the same mechanisms apply both to the germline and intestine cells?
Shift of SPD-2 coiled-coil protein is visible both in germline and intestine cells but SPD-2 is
present as foci in late pachytene nuclei and seems to be diffused in intestinal nuclei. So, we can
wonder, since these two kinds of cells undergo different cell cycle changes (meiosis and
endoreplicative cycle, respectively), if SPD-2 shift results from the same mechanism in both cases.
Investigation of SPD-2 partners could help to the comprehension of each phenomena. Then,
on the one hand, colocalization of each partner with SPD-2 by immunofluorescence experiments,
with confocal microscope, on the other hand, experiments investigating the interaction between
SPD-2 and Cyclin E/Cdk2, could lead to establish models of mechanism in each case.
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MATERIAL & METHODS

Nematodes strains
The following C. elegans strains were used: N2 Bristol was used as the wild type throughout.
MR1163 (unc119 (ed3) III; [spd-2::spd-2::GFP; unc-119(+)], from Roy lab); OC92 ([pCK6.1: pie1::GFP::spd-2; unc119(+)] , Kemp et al. 2004); unc-119 (ed3); WH163 (nDf29/unc-13(e1091)
spd-2(oj29) I), O'Connel et al. 1998). All C. elegans strains were cultured using standard techniques
and maintained at 15°C unless stated otherwise (Brenner, 1974).

Immunofluorescence microscopy
For Immunofluorescence labeling, worms were dissected onto gelatin coated microscope slides in
10l PBST (PBS containing 0.1% Tween-20). They were then freeze-cracked in dry ice for 10 min
before fixing in methanol at -20C for 1 min and post-fixing in formaldehyde solution for 30 min at
room temperature. Washing slides three times for 3 min in PBST blocked non-specific sites.
Primary antibodies (either rabbit polyclonal anti- SPD-2, a gift from K. O’Connell, National
Institutes of Health, Bethesda MD or mouse monoclonal anti- GFP) were applied at 1 g/ml in
PBST overnight at 4°C before washing 3 times for 10 min in PBST.
Secondary antibodies
(polyclonal anti-rabbit/mouse) were applied at 1 g/ml in PBST for 90 min at RT; the samples were
washed as above before mounting. DAPI was applied at 0.7 g/ml for 1 min, before washing the
slide in PBST for 1 min and adding 8 l Vector shield® on the slide. Indirect immunofluorescence
microscopy was performed using a 60× oil-immersion objective lens in a compound microscope
(DMR; Leica) equipped with a digital camera (C4742-95; Hamamatsu), imaging a μm-thick optical
section. Images were processed using Photoshop 8.0 (Adobe). All microscopic works were
performed at 20°C.

Western Blot assay
Protein extraction: For protein extraction, worms were immersed into H20 in presence of 5X
protein loading buffer, before shifting the temperature (from -80°C for 15 min, to 95°C for 5 min)
five times. The samples were then centrifuged at 13,500 rpm for 4 min and 5μl from the supernatant
were used as protein extract.
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Western Blot: Each protein extract was dissociated on SDS-PAGE (10%) and transferred onto
nitrocellulose membrane for 1 hour at 100V. Nitrocellulose membranes were then firstly incubated
in appropriate dilutions of primary rabbit polyclonal antibodies (anti SPD-2, at 1:2000 or anti GFP,
at 1:1000 in TBS 1X containing 5% milk) and primary mouse polyclonal antibodies (anti tubulin,) for 4h and then with secondary antibodies (Alkaline Phosphatase-conjugated antirabbit/mouse antibodies) for 1h. Chromogenic reaction was performed with Alkaline phosphatase
(AP) staining kit (Sigma). Fluorescence was finally analysed by scanner machine.

High-throughput in vitro site-directed mutagenesis (GeneTailor™ Site-directed
Mutagenesis System, Invitrogen™)
Target Plasmid: pMR811 is a 8 kb plasmid made from the pSK vector which contains the spd-2
gene promoter :: spd-2 genomic DNA sequence without the STOP codon and the last intron. 2.4 kb
upstream of the ATG of the spd-2 gene was selected as the promoter.
Control plasmid: The control plasmid is a 3.4 kb plasmid containing the lacZα gene, that when
introduced into wild type E. coli produces blue colonies on plates containing LB/Amp/X-gal.
1) Methylation Reaction: 125ng of target plasmid DNA where methylated with DNA methylase
for 1 hour at 37°C.
2) Amplification: The plasmid was amplified in a PCR mutagenesis reaction containing H20 (up to
50l) Phusion™ HF Buffer (1x), dNTPs (200M each), primers (0,5M each), target or control
DNA plasmid template (12.5ng) and the Phusion™ High-Fidelity DNA polymerase (0,02U/l)
Cycle Step
Temperature (°C)
Time
Cycles
Initial denaturation
98
30 s
1
Denaturation
98
8s
Annealing
74/ 722
20 s
Extension
72
3 min/ 45 s2
72
8 min
4
hold
Final extension
1
2
Best efficiency for mutagenesis PCR.
PCR cycling program for control plasmid and control primers.
201
1
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Master 1 ENS LYON
Two overlapping primers (one of which contains the target mutation), designed according to
Invitrogen™ Instruction manual:
Primer sequences
Tm (°C) 3
5'-ATTCCACAAATCACGACGAGAAAACGAGCGTACCTAAAAGAC-3' 4
79.60
5'- CTCGTCGTATTTGTGGAATTCATCGGTGA-3' 4
74.70
5'-GACCATGATTACGCCAAGCTTATAAATTAACCCT -3' 5
71,90
5'-AGCTTGGCGTAATCATGGTCATAGCTGTTT -3' 5
73,20
3
Tm calculation was made using the nearest-neighbor method, on www.finnzymes.com
4
Overlapping primers, red: overlapping region (20nt); black: extended region (10nt); green: mutation site
5
Control primers used with control plasmid and supplied with the GeneTailor™ mutagenesis System: The forward
(mutagenic) primer contains a two-base substitution that introduces a Hind III site and stop codon within the lacZα
gene. The stop codon generates a truncated LacZα protein and produces white colonies on plates containing X-gal
3) Transformation:
The linear double-stranded DNA product containing the mutation was
transformed into wild-type E.coli. The host cell circularized the linear mutated DNA and the
McrBC endonuclease present in the host cells, digested the methylated template DNA, leaving only
the unmethylated, mutated product.

RNAi:
As described previously (10), spd-2 RNAi was performed by feeding L1 worms with Escherichia
coli expressing spd-2 dsRNA.

Group-based phosphorylation scoring (GPS) method:
http://bioinformatics.lcd-ustc.org/gps_web/predict.php (12)

Gene bombardment:
The method was adapted from the protocol "DNA transformation by gene bombardment" which is
available on the website www.wormbook.org
BIBLIOGRAPHY
1. Delattre M. and Gonczy P. (2004) The arithmetic of centrosome biogenesis. Journal of
Cell Science.117; 1619-1629.
2. Boveri T. (1900). Zellen-Studien: Ueber die Natur der Centrosomen. Fisher, Jena,
Germany. 220 pp elegans. Nature. 434; 462–469.
Gehin Charlotte
Master 1 ENS LYON
3. Pelletier L. and al. (2006) Centriole assembly in C.elegans. Nature. 444
4. Leidel S. and Gonczy P. (2005) Centrosome duplication and nematodes: recent insights
from an old relationship. Dev. Cell. 9; 317-325.
5. Marshall WF. (2007) What is the function of centrioles? Journal of cell biochemistry. 100;
916-922.
6. Erich A. Nigg. (2007) Centrosome duplication: of rules and licenses. Trends in Cell Biology. 17;
No.5.
7. Dae Young Kim and Richard Roy. (2006) Cell cycle regulators control centrosome elimination
during oogenesis in C. elegans. Journal of Cell Biology. 174; No 6; 751-757.
8. Catherine A. Kemp and al. (2004) Centrosome Maturation and Duplication in C. elegans
require the Coiled-Coil Protein SPD-2. Developmental Cell. 6; Issue 6; 511-523.
9. www.wormbook.org
10. Ravi S. Kamath and Julie Ahringer. (2003) Genome-wide RNAi screening in Caenorhabditis
elegans Methods; 30, Issue 4, 313-321
11. Yu Xue, Fengfeng Zhou, Minjie Zhu, Guoliang Chen, and Xuebiao Yao. (2005) GPS: a
comprehensive www server for phosphorylation sites prediction. Nucleic Acids Res. 1 ; 33(Web
Server issue):W184-7.
12. Pelletier L. et al. (2004) The Caenorhabditis elegans Centrosomal Protein SPD-2 Is Required
for both Pericentriolar Material Recruitment and Centriole Duplication. Current Biology. 14 ; 10,
863-873.
13. www.wormbase.org
14. Mawuenyega KG, Kaji H, Yamuchi Y, Shinkawa T, Saito H, Taoka M, Takahashi N,
Isobe T (2003) Large-scale identification of Caenorhabditis elegans proteins by multidimensional
liquid chromatography-tandem mass spectrometry. J Proteome Res.. 2(1):23-35.
15. 0'Connel KF. Et al.: (1998). A genetic screen for temperature-sensitive cell-division mutants of
Caenorhabditis elegans. Genetics 149: 1303-1321
16. Kemp C. et al. (2007) Suppressors of zyg-1 define regulators of centrosome duplication and
nuclear association in C. elegans. Genetics 176: 95-113
17. Harris JE. et al. (2006) Major sperm protein signaling promotes oocyte microtubule
reorganization prior to fertilization in Caenorhabditis elegans. Developmental biology 299: issue 1;
105-121
18. Delattre M., Canard C. annd Gönczy P. (2006) Sequential protein recruitment in C.elegans
centriole formation. Current biology 16: issue 18; 1844-1849
Gehin Charlotte
Master 1 ENS LYON
ANNEXES
Figure 8. The most probable Cdk-phosphorylation sites of SAS-4, SAS-5, SAS-6, ZYG-1
and SPD-2 sequences, as determined by the Group-based Phosphorylation Site (GPS)
scoring method (12). GPS scoring is a statistical method. Best scores correspond to most
probable phosphorylation sites. On the figure, small stars surmount the amino acids most
likely to be targeted by CDKs. Only scores higher than 4 were considered as significant.
Figure 9. Centrioles are gradually lost after karyokinesis in intestinal nuclei. Extruded intestine from N2, stained for SPD-2 (green)
and another centriole marker, SAS-6 (red) SPD-2 and SAS-6 colocalize in centriolar foci (stars) around nuclei (arrowheads) from the
first larval stages (L1-L2). However, this number of foci decreases throughout post-embryonic development (as shown in the table)
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Master 1 ENS LYON