An RNAi-based modifier screen for novel mitotic regulators
The one-cell-stage C. elegans
embryo is well suited for analyzing the onset of mitosis and the execution of NEBD, which can be monitored with exquisite spatial and temporal resolution (). Using embryos in which the male and female pronuclei remain apart due to defective pronuclear migration, we found previously that such separated pronuclei undergo asynchronous NEBD () in a manner that is dependent on centrosomes and on AIR-1 (Hachet et al., 2007
FIGURE 1: RNAi-based modifier screen for modulators of mitotic entry. (A and B) Images from time-lapse DIC microscopy of wild-type (A) and zyg-9(b244) (B) one-cell-stage embryos. In all figures, anterior is to the left, posterior to the right; F and M designate (more ...)
To uncover novel genes modulating mitotic entry, we used this assay to design an RNAi-based modifier screen to identify components contributing to the asynchrony normally observed when the two pronuclei are separated. We anticipated this screen to identify positive and negative regulators of mitotic entry. In principle, such regulators could act in a centrosome-dependent manner and thus exhibit alterations in the timing of the male pronucleus, which is the only one associated with centrosomes in this setting. Alternatively, such regulators may function in a centrosome-independent manner, in which case they might exhibit also, or perhaps only, alterations in the timing of the female pronucleus.
We selected a set of genes to screen using two criteria. First, we chose ~1400 genes that, based on a compendium of microarray experiments, are coexpressed with known mitotic regulators, including ncc-1
, and air-1
(Kim et al., 2001
). Second, we selected genes that are embryonic lethal when inactivated by RNAi, thus reducing the number of genes to test to 360 (Table S2). These 360 genes were inactivated singly by RNAi in the background of zyg-9(b244)
mutant embryos, which exhibit defective pronuclear migration and asynchronous NEBD (). In each case, initially three to five embryos were analyzed by time-lapse differential interference contrast (DIC) microscopy, and the average time difference between the two pronuclei was determined (). A subsequent confirmation round, during which more embryos were analyzed, was performed with candidate genes identified in the initial screen.
In this manner, we identified five genes whose inactivation clearly increases the asynchrony between the separated pronuclei (). These genes encode PLK-1, which was only partially inactivated by RNAi in this experiment (see Materials and Methods
), the Bora homologue SPAT-1, which regulates Aurora-A and functions with PLK-1 to regulate cell-cycle progression (Hutterer et al., 2006
; Noatynska et al., 2010
), two modulators of the Rho GTPase, the RhoGEF ECT-2 and the RhoGAP CYK-4, as well as the Aurora-B kinase AIR-2. The latter three genes are essential for cytokinesis, including during the meiotic divisions, and we hypothesize that the apparent increased asynchrony in these cases reflects delayed formation of the female pronucleus following impaired meiotic divisions. These five components are not the focus of this study and thus will not be discussed further.
We also identified eight genes whose inactivation clearly decreases the asynchrony between the separated male and female pronuclei (). The corresponding proteins include four that were expected from earlier work: AIR-1, SPD-2 and SPD-5 (both required for centrosome integrity [ Hamill et al., 2002
; Kemp et al., 2004
]), as well as SUN-1 [required to link centrosomes to the male pronucleus (Malone et al., 2003
)]. Another protein whose inactivation decreases asynchrony is RAN-3, the Ran GTPase GEF RCC1. Interestingly, the three remaining proteins in this category are the nucleoporins Nup205/NPP-3, Nup54/NPP-1, and Nup35/NPP-19, which in other systems are known to interact with each other (Grandi et al., 1995
; Kosova et al., 1999
; Hawryluk-Gara et al., 2005
These results led us to analyze systematically the impact on timely NEBD of depleting the nucleoporins NPP-1 to NPP-20, because some of them were not included in the initial set of 360 genes. The more recently described NPP-21 to NPP-23 were not analyzed. As shown in Figure S1 and Table S3, we found that the depletion of another nucleoporin, the Nup93 homologue NPP-13, which in other systems is also part of the same subcomplex as Nup205/NPP-3 (Kosova et al., 1999
; Hawryluk-Gara et al., 2005
), also significantly reduces the asynchrony between the two pronuclei. Moreover, we found that NE formation is severely compromised in npp-8(RNAi)
, and npp-20(RNAi)
embryos (Galy et al., 2003
), precluding a thorough analysis of a potential contribution to timely mitotic entry. The same is true of most npp-19
) embryos (Rodenas et al., 2009
), despite the fact that some embryos were analyzable in the initial screen, presumably owing to incomplete RNAi-mediated inactivation (see ).
Given that the depletion of NPP-3 has the most striking impact on asynchrony (), we focused further investigations on this component.
NPP-3 as a novel negative regulator of mitotic onset
Time-lapse DIC recordings of a larger number of embryos confirmed that the onset of NEBD is synchronous in zyg-9(b244) embryos following NPP-3 depletion (, and S1, Table S3, and Movies M1 and M2). We also assayed NEBD in live embryos using yellow fluorescent protein (YFP)-lamin to visualize the lamina underlying the NE. The onset of lamina disassembly is synchronous in the two pronuclei in the wild type, whereas it is asynchronous in zyg-9(RNAi) embryos (, Movie M3). As anticipated, we found that lamina disassembly is synchronous in the two pronuclei of zyg-9(RNAi) npp-3(RNAi) embryos (, and Movie M4).
FIGURE 2: NPP-3 depletion abolishes asynchronous NEBD of separated male and female pronuclei. (A and C) Images from time-lapse DIC microscopy of zyg-9(b244) (A) and zyg-9(b244) npp-3(RNAi) (C) one-cell-stage embryos. See also corresponding Movies M1 and M2. (E (more ...)
We next addressed whether the synchrony observed when scoring the onset of NEBD upon NPP-3 depletion reflects a more general impact on the timing of mitotic entry, in which case it should be accompanied by synchronous changes of Cdk1 activity in the two pronuclei. To address this question, we used a Cdk1P-Tyr15
antibody that recognizes specifically the inactive form of the NCC-1 kinase. As reported previously (Hachet et al., 2007
), when NCC-1 is inactive during interphase, the Cdk1P-Tyr15
signal is high in both separated pronuclei. During prophase, the Cdk1P-Tyr15
signal diminishes earlier in the male pronucleus than in the female pronucleus in the majority of zyg-9(b244)
embryos, indicative of earlier Cdk1 activation in the male pronucleus (; 76%, N
= 25 embryos) (Hachet et al., 2007
). As shown in , we found by contrast that the Cdk1P-Tyr15
signal diminishes earlier in the male pronucleus only in a minority of zyg-9(b244) npp-3
) embryos (; 43%, N
= 42 embryos, binomial two-tail test calculation, p
= 3.88 × 10−7
). This result suggests that NPP-3 depletion has a more general effect on the timing of mitotic entry, although the impact on the onset of NEBD is most readily detectable.
In principle, synchronous mitotic entry in zyg-9(b244) npp-3(RNAi)
embryos could be due to a delay of the male pronucleus or, instead, to an acceleration of the female pronucleus compared with zyg-9(b244)
alone. To distinguish between these possibilities, we determined the overall duration of the first cell cycle by monitoring live embryos since the exit from meiosis II. Previous work established that the female pronucleus is delayed compared with the wild type in zyg-9(b244)
embryos (Hachet et al., 2007
). Here we found that the two separated pronuclei in zyg-9(b244) npp-3(RNAi
) embryos enter mitosis approximately at the same time as do the two joined pronuclei in the wild type (). Therefore the synchrony in zyg-9(b244) npp-3(RNAi)
embryos reflects an acceleration of mitotic entry in the female pronucleus, which is devoid of centrosomes in this setting.
These findings led us to conclude that NPP-3 acts as a negative regulator of mitotic onset in zyg-9
mutant embryos, the contribution of which is masked by the presence of centrosomes next to the male pronucleus. We then addressed whether NPP-3 depletion also accelerates mitotic onset in embryos lacking centrosomes, in which both centrosomes enter mitosis with wild-type timing (Hachet et al., 2007
). To this end, we analyzed the overall duration of the first cell cycle in spd-5(or213) npp-3(RNAi)
embryos, and found it to be statistically indistinguishable from that in spd-5(or213)
embryos (mean, SD, and N:
940, 46, 10 vs. 920, 38, 13; p
= 0.51, Student's t
test). This finding indicates that NPP-3 depletion cannot advance mitotic onset further, presumably because another rate-limiting component is not active as of yet.
Nuclear permeability defects do not correlate with decreased asynchrony
We considered whether general permeability defects in the NE could explain synchronous entry into mitosis in zyg-9(b244) npp-3(RNAi)
embryos. Therefore we monitored nuclear permeability defects by monitoring the ability of pronuclei to exclude fluorescently labeled 70- and 155-kDa dextrans. Consistent with previous observations (Galy et al., 2003
), we found that the size exclusion limit of the nucleus is defective in npp-3
) embryos, because 70-kDa dextran is not excluded from the pronuclei, whereas 155-kDa dextran is correctly excluded ().
FIGURE 3: Defective nuclear permeability does not drastically alter timing of mitotic entry. (A–C) Nuclear exclusion of fluorescently labeled dextrans of 70 kDa (green) and 155 kDa (red). Confocal images of wild-type (A), npp-3(RNAi) (B), and npp-4(RNAi) (more ...)
Although this result could be compatible with general NE permeability defects being responsible for accelerated entry into mitosis of the female pronucleus in zyg-9(b244) npp-3(RNAi) embryos, we addressed this possibility further by investigating whether NE permeability defects always cause synchrony. To this end, we investigated the exclusion from the pronuclei of 70- and 155-kDa dextrans in embryos depleted of other nucleoporins. As shown in and S2, A–E, we found that embryos depleted of Nup93/NPP-13 or Nup45/58/NPP-4 exhibit nuclear permeability defects indistinguishable from those in embryos depleted of NPP-3. Despite this, zyg-9(b244) npp-13(RNAi) [and even more so, zyg-9(b244) npp-4(RNAi)] embryos retain substantial asynchrony between the two pronuclei ( and Table S3). As a further means to compare permeability defects in npp-3(RNAi) and npp-4(RNAi) embryos, we monitored the ability of pronuclei to exclude green fluorescent protein (GFP)-β-tubulin, which was similarly compromised in embryos depleted of either NPP-3 or NPP-4, as well as of NPP-1 (Figure S2, F–I). Analogous conclusions were reached when comparing the distribution of GFP-β-tubulin upon NPP-3 and NPP-4 depletion in zyg-9(b244) embryos (Movies 5–7).
Overall, these findings indicate that bulk permeability defects, although potentially being a contributing factor, do not alone explain synchronous onset of NEBD and mitotic entry in zyg-9(b244) npp-3(RNAi) embryos. Instead, we postulate that NPP-3 can negatively impact timely mitotic entry when centrosomes are not in the vicinity of the NE by ensuring the proper nuclear/cytoplasmic distribution of a specific cell-cycle regulator. Upon NPP-3 depletion in a zyg-9(b244) mutant background, this as-of-yet unidentified component may gain premature access inside the female pronucleus and thus lead to synchronous mitotic entry of the two pronuclei.
Decreased asynchrony correlates with diminished NPP-3 at the NE
We proceeded to analyze the subcellular distribution of NPP-3. We raised and affinity-purified polyclonal antibodies against NPP-3, which recognize a single specific band at the expected size in wild-type embryonic extracts (). Immunofluorescence analysis of wild-type, one-cell-stage embryos probed with these antibodies demonstrates that NPP-3 localizes primarily at the NE ().
FIGURE 4: Decreased asynchrony correlates with loss of NPP-3 at the NE. (A) Western blot of extracts from wild-type or npp-3(RNAi) embryos probed with NPP-3 antibodies. (B–I) NPP-3 distribution at the NE in embryos of the indicated genotypes stained for (more ...)
We set out to investigate whether alterations in NPP-3 distribution at the NE could explain the partial loss of asynchrony observed upon depletion of other nucleoporins in zyg-9(b244) embryos. We observed a strong reduction of NPP-3 at the NE upon depletion of Nup93/NPP-13 or of Nup54/NPP-1, the two NPPs that had the most significant impact on asynchrony after NPP-3 itself (, and Table S4). In contrast, we found that depletion of Nup45/Nup58/NPP-4, Nup160/NPP-6, Nup153/NPP-7, or Nup85/NPP-2, which all have a minor impact on asynchrony, does not strikingly alter NPP-3 levels at the NE (, and Table S4).
Overall, these findings establish that lower levels of NPP-3 at the NE correlate with accelerated entry into mitosis in the female pronucleus in zyg-9(b244) embryos, indicating that the presence of NPP-3 at the NE negatively impacts mitotic entry.
NPP-3 local removal from the NE at mitosis
While investigating the distribution of NPP-3 across the cell cycle, we discovered that the protein is no longer detected at the NE in the vicinity of centrosomes, starting at the end of prophase and most strikingly in prometaphase (). This is unlikely because of epitope masking, because polyclonal antibodies raised against a fusion protein were used. Moreover, a similar local loss is observed with antibodies against NPPs that are part of the same subcomplex (see later in the text). The lamina has been reported to disassemble initially in the vicinity of centrosomes in C. elegans
embryos (Lee et al., 2000
). By conducting double-labeling experiments, we found that NPP-3 local loss precedes lamina disassembly (). We conclude that NPP-3 local loss from the NE is an early event of NEBD.
FIGURE 5: NPP-3 localization at mitotic onset. (A–D) Wild-type, one-cell-stage embryos in prophase (A and B), prometaphase (C), or metaphase (D) stained for NPP-3 (shown alone in the insets on the right and in red in the merged images), α-tubulin (more ...)
We next investigated whether such local loss is also exhibited by other nucleoporins. We first tested whether this is the case for the NPP-3–associated Nup93/NPP-13. To this end, we raised and affinity-purified polyclonal antibodies against NPP-13, which decorate the NE of wild-type embryos (Figure S3, A–D). Importantly, we found that NPP-13 also exhibits local loss near centrosomes at the onset of mitosis, concomitant with that of NPP-3 (). Similar results were obtained with embryos expressing GFP–NPP-19 (Figure S3, E and F). In contrast, we found that Nup107/NPP-5, which resides on the outer part of the NPC, as well as Nup98/NPP-10 and Nup153/NPP-7, which reside on the inner part of the NE, all disappear in a uniform manner from the NE and redistribute to kinetochores in mitosis (, Figure S3, G and H, and data not shown; Belgareh et al., 2001
; Rodenas et al., 2009
). To investigate whether the overall integrity of the NE is compromised at the time of NPP-3 local loss, we analyzed the distribution of the inner nuclear membrane components GFP-emerin and GFP–LEM-2, as well as that of the GFP-SP12 fusion that marks ER-localized proteins and thus also labels the outer nuclear membrane (Lee et al., 2000
; Poteryaev et al., 2005
). We found that these three fusion proteins do not exhibit local loss at the onset of mitosis (Figure S3I and data not shown), indicating that NPP-3 local removal does not result from an overall loss of membrane integrity.
Overall, we conclude that NPP-3, NPP-13, and NPP-19, which are all members of the Nup93 subcomplex, exhibit local loss from the NE in the vicinity of centrosomes at the onset of mitosis.
Centrosomes and AIR-1 kinase activity direct NPP-3 local removal from the NE
We set out to address the mechanisms that result in local loss of NPP-3 and associated NPPs from the NE in the vicinity of centrosomes at the onset of mitosis. To address whether centrosomes are necessary, we disrupted centrosome integrity through depletion of SPD-5 and found that this prevents NPP-3 local loss (). To address whether centrosomes are sufficient, we depleted ZYG-12 such that the two centrosomes detach from the male pronucleus early in the cell cycle and move freely in the cytoplasm until they each contact stochastically one of the two separated pronuclei. We found that NPP-3 is invariably lost from the NE in the vicinity of the centrosome in such embryos (). We conclude that centrosomes are both necessary and sufficient for NPP-3 local loss from the NE at mitotic onset.
FIGURE 6: NPP-3 local loss is centrosome- and AIR-1–dependent. (A–J) One-cell-stage embryos of the indicated genotypes stained for NPP-3 (shown alone in the insets on the right and in red in the merged images), α-tubulin (green), and DNA (more ...)
We next investigated whether microtubules nucleated from the centrosomes are involved. As shown in , we found that NPP-3 local loss is not altered upon depletion of the γ-tubulin component TBG-1, which significantly decreases centrosomal microtubules (Hannak et al., 2002
). Moreover, NPP-3 local loss is also unaffected when microtubules are essentially absent following RNAi-mediated depletion of the α-tubulin gene tba-2
(). The same is true of embryos simultaneously depleted of ZYG-12 and TBA-2 function, in which residual short microtubules that could remain on tba-2
) alone are expected not to contact the NE (). We conclude that centrosomes appear to dictate NPP-3 local loss independently of microtubules.
We then verified whether NCC-1, which is known to be essential for NEBD and mitotic entry (Boxem et al., 1999
), affects NPP-3 local loss, and found this to be the case indeed (). Next we tested whether the centrosomal kinase AIR-1, the depletion of which also leads to synchronous NEBD of separated pronuclei (Hachet et al., 2007
; Portier et al., 2007
), regulates NPP-3 local loss. As shown in , we found that NPP-3 local loss does not occur in air-1
) embryos. To address whether AIR-1 kinase activity is needed, we analyzed embryos depleted of endogenous air-1
and expressing an RNAi-resistant transgene encoding either wild type or a kinase-inactive version of GFP-tagged AIR-1. As illustrated in , we found that NPP-3 local loss occurs in embryos expressing wild-type AIR-1, but not the corresponding kinase-inactive version.
These results demonstrate that centrosomes and AIR-1 kinase activity dictate local loss of NPP-3 from the NE at the onset of mitosis. Together with our finding that NPP-3 can negatively regulate the timing of mitotic onset, these findings lead us to propose a model whereby centrosomes and AIR-1 promote timely onset of mitosis by leading to the local removal of NPP-3 and associated nucleoporins from the NE ().
FIGURE 7: Model for the role of centrosomes and AIR-1 in promoting NPP-3 local loss and timely mitotic entry in C. elegans embryos. Green circle: AIR-1 at centrosomes. Note that cytoplasmic AIR-1 is not depicted in this figure but likely contributes to overall (more ...)