SZY-20 is a negative regulator of centrosome duplication
gene was identified in a screen for genetic suppressors of the temperature-sensitive (ts) lethality of the zyg-1(it25)
mutation (Kemp et al., 2007
). In zyg-1
embryos raised at the restrictive temperature (24°C), duplication of the sperm-derived centriole pair invariably fails. This initial centriole pair, however, is able to separate and direct formation of a bipolar spindle. Such embryos divide once to form a two-cell embryo but during the second round of mitosis, each blastomere assembles a monopolar spindle (). As a result, 100% of the embryos die (O'Connell et al., 2001
). The szy-20(bs52)
mutation however suppresses the embryonic lethality of zyg-1(it25)
such that a percentage of double mutant embryos are able to complete development and hatch at the restrictive temperature (). Importantly, the szy-20(bs52)
mutation is unable to suppress a complete loss of zyg-1
activity indicating that suppression might involve upregulation of ZYG-1 (Kemp et al., 2007
Figure 1 Loss of szy-20 activity suppresses centrosome duplication defects in zyg-1 and spd-2 mutants. (A–C) Centrosome duplication and bipolar spindle formation are restored in zyg-1(it25) szy-20(bs52) embryos. (A) Selected images from 4D-DIC recordings. (more ...)
To investigate the basis of suppression, we analyzed bipolar spindle formation and centrosome duplication directly in zyg-1
double mutant embryos. By four-dimensional differential interference contrast (4D-DIC) imaging ( and Movie S1
), we found that zyg-1(it25) szy-20(bs52)
embryos are often successful in assembling bipolar spindles at the two-cell stage (13/24 events), unlike zyg-1(it25)
embryos (0/28 events). The ability of the double mutant to assemble bipolar spindles appears to result from suppression of the centrosome duplication defect, as spindle poles in zyg-1(it25) szy-20(bs52)
embryos invariably stained with the centriole marker SAS-4 (). The ability of the szy-20(bs52)
mutation to suppress the centrosome duplication defect was further confirmed by fluorescence imaging of live embryos expressing GFP-SPD-2 to mark centrosomes and GFP-histone to mark DNA ( and Movie S2
). As szy-20(bs52)
is a loss-of-function allele (see below), our results demonstrate that szy-20
negatively regulates centriole assembly.
The szy-20(bs52) mutation restores centrosome duplication in spd-2 mutants
We next asked if the szy-20(bs52)
mutation could also suppress mutations in other genes required for centrosome duplication. spd-2
is required for both centrosome duplication and PCM assembly. In spd-2
mutants, the sperm-derived centriole pair fails to duplicate and only weakly organizes microtubules (Kemp et al., 2004
; Pelletier et al., 2004
). As a result, spd-2
mutants fail to assemble the first mitotic spindle and arrest at the one-cell stage. We generated a double mutant line and found that the szy-20(bs52)
mutation only weakly suppresses the embryonic lethality of the hypomorphic spd-2(or188)
mutation (data not shown). When we examined the spd-2(or188); szy-20(bs52)
double mutant by immunofluorescence microscopy, we found that the microtubule organization and one-cell arrest phenotypes were still present. However, as judged by SAS-4 immunostaining, the double mutant embryos exhibited evidence of centrosome duplication (). While only seven percent of one-cell spd-2(or188)
embryos (n=28) possessed more than the two original sperm-derived centrioles, 65% of spd-2(or188); szy-20(bs52)
embryos (n=45) possessed three or more centrioles. Thus, szy-20
genetically interacts with zyg-1
, two genes whose products function at the earliest known step in centrosome duplication (Delattre et al., 2006
; Pelletier et al., 2006
As a control we assayed the ability of the szy-20(bs52)
mutation to suppress an unrelated ts mutant. At 24°C, the mat-3(or180)
mutation blocks the function of the anaphase-promoting complex, producing an embryonic lethal phenotype marked by a one-cell arrest (Stein et al., 2007
). Analysis of a szy-20(bs52); mat-3(or180)
double mutant revealed that szy-20(bs52)
suppresses neither the embryonic lethal phenotype nor the one-cell arrest of mat-3(or180)
mutants (data not shown), indicating that szy-20(bs52)
is not a general suppressor of ts mutations.
Maternally supplied SZY-20 is essential for embryonic development
mutation confers its own recessive ts embryonic lethal phenotype ( and Kemp et al., 2007
). At 20°C greater than 50% of the progeny of szy-20(bs52)
hermaphrodites are viable, but at 25°C nearly all of the progeny fail to hatch. As the embryonic lethality of szy-20(bs52)
hermaphrodites can not be rescued by mating to wild-type males (data not shown), maternally-expressed SZY-20 is essential for embryogenesis.
Preliminary analysis of szy-20(bs52)
embryos revealed defects in cytokinesis and in attachment of the centrosome to the nucleus (Kemp et al., 2007
). To further characterize the consequences of loss of szy-20
, we analyzed a large number of szy-20(bs52)
embryos stained for microtubules, DNA and centrosomes (). At 25°C, szy-20(bs52)
embryos exhibit multiple defects in cell division. As observed previously, some embryos contain extra nuclei and centrosomes, a phenotype suggestive of cytokinesis failure (20%, n=135; ), while in others, centrosomes lose contact with the nuclear envelope (12%, n=135; ). We also observed defects that had not been previously observed. szy-20(bs52)
embryos exhibit a 10% decrease in the length of metaphase spindles compared to wild-type embryos (n=12; ). We also noticed the presence of more than one sperm pronucleus-centrosome pair in a small fraction (<5%) of embryos, a phenotype suggestive of polyspermy ().
Figure 2 SZY-20 is required for cell division. szy-20(bs52) embryos stained for microtubules (green), centrosomes (red) and DNA (blue). (A) A one-cell embryo with four centrosomes stained for ZYG-1. (B) A two-cell embryo with detached centrosomes (arrows) stained (more ...)
Figure 4 SZY-20 limits the size and the microtubule-nucleating capacity of the centrosome. (A) Embryos at first metaphase stained for SPD-5 (red) and DNA (blue). (B) Fluorescence intensity measurements of centrosomal and cytoplasmic SPD-5. Vertical bars indicate (more ...)
To further define szy-20
function, we performed live imaging of szy-20(bs52)
embryos grown at 25°C (Movie S3
). Time-lapse recordings confirmed a cytokinesis defect in szy-20(bs52)
embryos. In these embryos, the furrow initiates normally but fails to ingress completely, resulting in the formation of a multinucleated cell with four centrosomes. szy-20(bs52)
embryos also frequently fail to extrude polar bodies and exhibit a slight (1.2–1.4 fold) lengthening of the cell cycle. While these defects are observed at all temperatures, the severity and penetrance of each defect increases at higher temperature. We also analyzed the deletion allele szy-20(tm1997)
and found that it confers a ts embryonic lethal phenotype and many of the cytological defects described for szy-20(bs52)
( and data not shown).
Molecular characterization of szy-20
To further define the role of SZY-20, we sought to clone the corresponding gene located between dpy-10
on chromosome II (Kemp et al., 2007
). Using a physical mapping approach, we localized szy-20
to a small interval of 54.2 Kb that encompasses 14 predicted open reading frames (ORFs; Figure S1
). Sequencing detected a nonsense (C-to-T) mutation in exon 6 of ORF C18E9.3 that presumably results in truncation of the gene product ( and S1
Figure 3 szy-20 encodes a novel conserved protein. (A) Schematic of wild-type and mutant forms of SZY-20. Due to a frame shift, the product of the szy-20(tm1997) allele is expected to contain a novel 36-amino acid extension (blue box). (B) Immunoblot of wild-type (more ...)
To confirm the identity of szy-20, we used RNAi to inhibit expression of C18E9.3 in both wild-type and zyg-1(it25) animals. In all respects, C18E9.3 RNAi phenocopied the szy-20(bs52) mutation. C18E9.3 RNAi partially suppressed zyg-1(it25) embryonic lethality (); this was most obvious at 23.5°C where C18E9.3 RNAi of zyg-1(it25) animals led to a 100-fold increase in the frequency of viable progeny (27.2 % versus 0.27% in controls). C18E9.3 RNAi also suppressed the embryonic lethality of the zyg-1(or409) allele. In wild-type animals, C18E9.3 RNAi produced a ts embryonic lethal phenotype reminiscent of szy-20(bs52): <10% embryonic lethality at 20°C, but ~90% at 25°C (). Further, combining the mutation and RNAi had a synergistic effect at 20°C: only ~5% of the progeny of szy-20(bs52) C18E9.3 RNAi mothers survived, compared to 55% for szy-20(bs52) and 92.3 % for C18E9.3 RNAi. Finally, szy-20(bs52) failed to complement szy-20(tm1997), a partial deletion of C18E9.3 (). We conclude that szy-20 corresponds to ORF C18E9.3. Based on the ability to phenocopy szy-20(bs52) by RNAi as well as the failure of szy-20(bs52) to complement mnDf68, a chromosomal deficiency that removes the szy-20 locus (), we conclude that szy-20(bs52) is a loss-of-function allele.
) lists multiple ESTs for C18E9.3. We sequenced four of these cDNA clones and identified two alternatively spliced transcripts (Figure S1
). The longer transcript encodes a protein of 558 amino acids (Isoform A; GenBank accession EF656060) and the shorter transcript a protein of 457 amino acids (Isoform B; GenBank accession EF656061). Both messages are trans
-spliced to the SL1 leader sequence and appear to utilize the same initiation and termination codons.
szy-20 encodes a conserved protein
A BLAST search (Experimental Procedures) identified SZY-20 orthologs in other species including flies and humans. All animal genomes with significant sequence data contain one ortholog of SZY-20, indicating a potentially conserved role across animals. A multiple alignment of SZY-20 with its homologs showed that they all share three prominent blocks of conservation ( and S2
). The N-terminal block is a short element with a characteristic hx[DE][SN]W[DE][DE] signature (where h is any hydrophobic residue and x is any residue), which is found exclusively in orthologs of SZY-20 (Figure S2A
). The second block in the center of the protein, termed the SUZ domain, is the strongest stretch of conservation and is enriched in charged residues (Figure S2B
). The third block, the SUZ-C domain, is at the extreme C-terminus and is defined by a characteristic pattern of two highly conserved glycines and one absolutely conserved proline (Figure S2C
). In nematodes, however, this domain has rapidly diverged.
We noticed that the SUZ and SUZ-C domains occur independently in proteins outside of the SZY-20 family (Figure S3 and Supplemental Results
). Interestingly, many of these proteins contain known RNA-binding domains suggesting that SZY-20 might function in RNA metabolism. We tested SZY-20 for RNA-binding in vitro
. GST-SZY-20 specifically and reproducibly co-precipitates with polyuridine-coupled beads, indicating that SZY-20 can directly bind RNA (Figure S4A
). Both the SUZ and SUZ-C domains contribute to this activity as mutation of either domain significantly reduces the ability of the protein to co-precipitate with the beads. Furthermore, RNA-binding is nearly eliminated in a double mutant (GST-SZY-20dm
) that carries mutations in both domains (Figure S4B
). To investigate the function of these putative RNA-binding domains in vivo
, we expressed FLAG-tagged versions of wild-type and SZY-20dm
in worms (Figure S4C
). When expressed in szy-20(bs52)
animals grown at 25°C, the wild-type FLAG-SZY-20 protein provided strong rescue of the embryonic lethality, reducing it to 52% (n=2615) from 96% in controls (n=936). In contrast, the FLAG-SZY-20dm
mutant had little effect (84% embryonic lethality, n=1215), consistent with the notion that RNA-binding is involved in SZY-20 function.
SZY-20 localizes to nuclei, cytoplasm and centrosomes
To determine how SZY-20 might function, we examined its subcellular distribution using affinity-purified antibodies. On immunoblots, the αS20N antibody detected a single band in wild-type embryonic extracts. The migration of this band was consistent with the size of isoform A (). This band was absent in szy-20(bs52) embryonic extracts. Instead a faster migrating band, presumably corresponding to the truncated product, was detected.
In immunostained embryos, SZY-20 is found in small foci in the cytoplasm and at centrosomes where it coincides with GFP-SAS-4, a marker of centrioles (). In addition, SZY-20 localizes to nucleoli during interphase and to the surface of chromosomes during meiosis and mitosis (Figure S5
). RNAi of szy-20
significantly reduces staining of all structures, confirming the specificity of the antibody (). SZY-20 is first detected at centrosomes during meiosis where it often appears as two distinct dots adjacent to the male pronucleus (). While SZY-20 localizes to the centrosome throughout the cell cycle, the extent of its association is regulated in a cell cycle-dependent manner (). The level of centrosome-associated SZY-20 peaks at prometaphase and metaphase, slowly declines, and reaches a minimal level during interphase. In contrast, the level of ZYG-1 at centrosomes is lowest at metaphase (Delattre et al., 2006
), the time when the level of SZY-20 peaks. ZYG-1 then rapidly increases during anaphase while SZY-20 levels drop. This reciprocal relationship between centrosomal levels of SZY-20 and ZYG-1 suggests that SZY-20 acts locally to regulate ZYG-1.
SZY-20 limits centrosome size
During our cytological characterization, we noticed that centrosomes appear larger in szy-20(bs52) than in wild-type embryos. This difference was apparent in embryos stained for the pericentriolar component SPD-5 (). We measured the fluorescence intensity of stained centrosomes at first metaphase () and found that szy-20(bs52) embryos (n=31) contain more than twice as much centrosomal SPD-5 as controls (n=10). In contrast, cytoplasmic pools of SPD-5 appear similar in the two strains, suggesting that the overall levels of SPD-5 are unaffected by the szy-20(bs52) mutation. This was confirmed by quantitative immunoblotting (), which showed that wild-type and mutant embryos possessed similar amounts of SPD-5 (mutant/wt = 1.12 ± 0.09, n=4). This result suggests that the szy-20(bs52) mutation might enhance recruitment of SPD-5 to centrosomes.
SPD-2 and SPD-5 function at the top of a hierarchy in the centrosome maturation pathway (Hamill et al., 2002
; Kemp et al., 2004
; Pelletier et al., 2004
). In addition to recruiting downstream components, SPD-2 and SPD-5 localize to PCM in a mutually dependent manner. We postulated that the szy-20(bs52)
mutation would affect the level of SPD-2 at centrosomes and analyzed embryos expressing GFP-SPD-2 (). As predicted, the level of centrosome-associated GFP-SPD-2 is significantly increased in the szy-20(bs52)
mutant at all stages of the cell cycle. The increase in centrosomal levels of GFP-SPD-2 reflects increases in both density (1.4 ± 0.6 fold) and cross-sectional area (1.4 ± 0.5 fold). However, we detected no significant difference in overall levels of endogenous SPD-2 between wild-type and szy-20(bs52)
embryos (mutant/wt = 1.08 ± 0.14, n=5) as shown in . Thus, the elevated levels of SPD-5 and SPD-2 at mutant centrosomes most likely result from enhanced recruitment.
To further confirm the role of SZY-20 in regulating centrosome size, we overexpressed SZY-20 in wild-type embryos (). Consistent with a role as a negative regulator, overexpression of wild-type FLAG-SZY-20 reduces the centrosomal level of GFP-SPD-2 by half (0.48 ± 0.1 fold, n=11) compared to controls (n=14). Interestingly, we did not observe an effect following overexpression of FLAG-SZY-20dm (1.2 ± 0.3 fold, n=12). Thus, the conserved domains that mediate RNA-binding in vitro are important for regulating centrosome size.
SZY-20 limits the microtubule-nucleating capacity of the centrosome
The microtubule-nucleating capacity of the centrosome positively correlates with the amount of PCM. Since centrosomes in szy-20(bs52) embryos contain elevated levels of PCM components, we wondered if such centrosomes nucleate more microtubules. First we analyzed the centrosome level of γ-tubulin, a PCM component directly involved in microtubule nucleation (). Similar to other centrosome proteins, γ-tubulin levels at centrosomes are elevated in the mutant (2.5 ± 1.2 fold, n=14) relative to the wild type (n=13).
We next investigated microtubule nucleation using strains expressing GFP-EBP-2, a marker for microtubule plus ends (Srayko et al., 2005
). Compared to controls, szy-20(bs52)
centrosomes exhibit a significant increase in GFP-EBP-2 fluorescence (). We then made time-lapse recordings of embryos expressing GFP-EBP-2. In projected images from these recordings, it was evident that more microtubules grow out from centrosomes in szy-20(bs52)
than in wild-type embryos ( and S6A
, Movie S5
). As a means of quantifying the difference in the outgrowth of microtubules between the two strains, we measured the intensity of GFP-EBP-2 in an area adjacent to the centrosome. We found that GFP-EBP-2 fluorescence is increased in the mutant (2.0 ± 0.6 fold, n=8) compared to controls (n=8), consistent with the presence of more centrosome-associated microtubules in the mutant. Interestingly, microtubule growth appeared stunted in the mutant. In a ten-second interval, many microtubules nucleated by wild-type centrosomes grow out to meet the cortex, while most microtubules nucleated by szy-20(bs52)
centrosomes grow only a short distance ( and S6B
, Movie S6
). In fact, only about half as many microtubules reach the cortex in szy-20(bs52)
embryos as in controls. The lack of long microtubules in the mutant is not due to insufficient recruitment of the microtubule-stabilizing protein ZYG-9 (Matthews et al. 1998
) as szy-20(bs52)
centrosomes (n=16) actually possess more ZYG-9 (1.7 ± 0.7 fold) than controls (n= 24) (). Instead, microtubules grow slower in the mutant (1.1 μm/s) than in the wild type (1.3 μm/s). Our results are reminiscent of those of (Srayko et al., 2005
) who found conditions that favor microtubule polymerization slow the growth rate of individual microtubules, presumably by depleting free tubulin subunits.
SZY-20 regulates the level of centrosome-associated ZYG-1
mutations, we wondered if the loss of SZY-20 affects the localization of ZYG-1. In immunostained szy-20(bs52)
embryos, we found that the level of ZYG-1 at the centrosome varies during the cell cycle as it does in the wild type, with the lowest level at metaphase and highest level at anaphase. However, at all cell cycle stages, the level of ZYG-1 is higher at szy-20(bs52)
centrosomes than at wild-type centrosomes ( and S7
). This is evident during prometaphase and metaphase where ZYG-1 was detected in 64% of szy-20(bs52)
centrosomes (n=117) but in only 23% of wild-type centrosomes (n=86). Quantitative fluorescence microscopy at metaphase revealed that szy-20(bs52)
mutants contain more than twice as much centrosome-associated ZYG-1 as wild-type embryos (). However, we found no difference in the cytoplasmic levels of ZYG-1 between the two strains, suggesting that loss of szy-20
activity does not simply result in overexpression of ZYG-1. This was confirmed by quantitative immunoblotting () which found that wild-type and mutant embryos possess equivalent amounts of ZYG-1 (mutant/wt = 1.03 ± 0.12, n=6). In contrast, we found that depletion of ZYG-1 did not affect SZY-20 localization (data not shown). Thus, SZY-20 regulates ZYG-1 levels at the centrosome but not vice-versa.
Figure 5 Loss of SZY-20 activity enhances ZYG-1 localization at centrosomes. (A) a Z-projections of embryos at first metaphase stained for microtubules (green), ZYG-1 (red) and DNA (blue). ZYG-1 is detectable only at szy-20(bs52) centrosomes. Bar, 5 μm. (more ...)
As the enlarged centrosomes in szy-20(bs52)
mutants possess elevated levels of the centriole duplication factor ZYG-1, we wanted to determine if they also possess centrioles of altered structure. Thin section TEM revealed that centrioles are of similar size and structure in wild-type (n=2) and szy-20(bs52)
centrosomes (n=10) ( and S8
). Thus, the elevated level of PCM observed in szy-20
mutants appears to occur in the absence of centriole duplication defects.
Centriole duplication factors regulate centrosome size
In the course of our analysis, we uncovered an unusual genetic relationship between zyg-1 and szy-20. Not only does szy-20(bs52) suppress zyg-1(it25) but the converse is also true (). At 20°C, nearly 100% of the progeny produced by zyg-1(it25) hermaphrodites are viable while only 55% of szy-20(bs52) embryos are viable. Remarkably, over 90% of the progeny of zyg-1(it25) szy-20(bs52) double mutants are viable. This difference between the szy-20(bs52) and zyg-1(it25) szy-20(bs52) strains is statistically significant (p <0.01), thus at 20°C loss of zyg-1 activity restores proper embryonic development to szy-20(bs52) mutants.
The mutual suppression exhibited by zyg-1
loss-of-function mutations suggests a simple model. A reduction in SZY-20 activity results in increased levels of ZYG-1 at the centrosome, which in turn has deleterious consequences for the embryo. Reducing ZYG-1 activity in the szy-20(bs52)
mutant could restore an appropriate level of centrosome-associated ZYG-1 and thus normal cellular processes. To analyze the interactions between zyg-1
at a cytological level, we performed time-lapse microscopy. At 20°C, szy-20(bs52)
zygotes exhibit highly penetrant defects in microtubule-dependent processes such as pronuclear rotation (87%, n=23; ) and cytokinesis (63%, n=19; Movie S3
). In wild-type embryos, the two apposed pronuclei rotate 90° to position the centrosomes and ultimately the spindle on the A–P axis. While this rotation is normally completed by prometaphase in wild-type embryos, it often does not initiate until metaphase in szy-20(bs52)
embryos. We observed robust rescue of both defects in szy-20(bs52)
mutants when zyg-1
activity was reduced. In the zyg-1(it25) szy-20(bs52)
double mutant, the pronuclear rotation defect is reduced to 12.5% (n=16) and the cytokinesis defect to 17% (n=12).
Figure 6 Centriole duplication factors regulate centrosome size. (A, B) Images from recordings of embryos expressing GFP-SPD-2 and GFP-histone. (A) Reducing zyg-1 activity restores normal centrosome size and function to szy-20(bs52) mutants. Note that the delay (more ...)
The pronuclear rotation and cytokinesis defects of szy-20(bs52) embryos could be a consequence of the enlarged centrosomes. That is, by nucleating more microtubules, the enlarged centrosomes deplete free tubulin and impede the formation of long microtubules that are needed for these processes. If true, suppression of both defects by the zyg-1(it25) allele might involve the restoration of centrosome size. To test this, we compared the level of centrosome-associated GFP-SPD-2 in szy-20(bs52) and zyg-1(it25) szy-20(bs52) zygotes grown at 20°C (). Consistent with our prediction, GFP-SPD-2 fluorescence was reduced by nearly half (0.59 ± 0.07) at zyg-1(it25) szy-20(bs52) centrosomes (n=12) compared to szy-20(bs52) centrosomes (n=14). Importantly, these experiments were conducted at 20°C where centrosome duplication is not blocked by the zyg-1(it25) mutation. Thus, the reduction in centrosome size is not a consequence of blocking daughter centriole formation. We further found that in the wild type, depletion of ZYG-1 by RNAi reduces the level of GFP-SPD-2 at the centrosome by more than half (0.4 ± 0.26 fold, n=11) relative to controls (n=10). Therefore, ZYG-1 has two separable functions. It regulates centrosome duplication and controls centrosome size. These results also show that inappropriately high levels of ZYG-1 at the centrosome can perturb centrosome function.
Finally, we investigated if the ability to regulate centrosome size is shared by other centriole duplication factors. We used RNAi to deplete SAS-6 in both wild-type and szy-20(bs52)
embryos expressing GFP-SPD-2 ( and S9
, Movie S7
). Compared to controls (n=20), depletion of SAS-6 in szy-20(bs52)
mutants reduced the amount of GFP-SPD-2 at centrosomes by nearly half (54%, n=23), an effect similar in magnitude to that produced by depleting ZYG-1 activity. In wild-type embryos, sas-6(RNAi)
(n=14) produced an even greater effect reducing the amount of centrosome-associated GFP-SPD-2 to 40% of controls (n=11). Interestingly, while sas-6(RNAi)
invariably blocked centrosome duplication in wild-type embryos, we found it to be much less potent in szy-20(bs52)
embryos where centrosomes were often able to duplicate (data not shown). This suggests that the szy-20(bs52)
mutation can also partially suppress the duplication defect in SAS-6 deficient embryos. Our results thus uncover a second function for ZYG-1, SAS-6, and possibly other centriole assembly factors: to regulate centrosome size.