Identification of Essential Ran and Ran-related Genes
To study the role of Ran in mitotic spindle formation and NE assembly, we identified C. elegans
homologues for Ran and Ran cofactors (RanGAP, RanBP2, and RCC1) based on published data (Gönczy et al., 2000
) and database searches (Table ; Bamba et al., 2002
). Because importin α and β have been demonstrated to play critical roles in spindle formation and/or NE assembly in Xenopus
extracts (Gruss et al., 2001
; Nachury et al., 2001
; Wiese et al., 2001
; Zhang et al., 2002
) we included the C. elegans
homologues in our studies. Geles and Adam (2001)
have described three C. elegans
homologues of importin α, IMA-1, -2, and -3, whereas importin β is homologous to C. elegans
IMB-1 (Geles and Adam, 2001
; Table ). Because RNAi against IMA-1 gives no observable phenotype (Geles and Adam, 2001
; our unpublished data) IMA-1 was not analyzed further.
RNAi effects on brood size and viability
As an initial characterization of the seven genes (Table ), we evaluated the effect of RNAi on brood size and embryo viability. We found that all seven genes are required for embryonic development because 91–100% of the embryos failed to hatch (Table ). For some of the genes this is in agreement with previous observations (IMA-2, Fraser et al., 2000
; Ran, RanGAP, and RanBP2, Gönczy et al., 2000
; and IMA-3, Geles and Adam, 2001
), whereas others had not been tested before. RNAi of Ran significantly reduced the brood size, indicating that Ran is also required at some step during oogenesis, whereas RNAi of any of the regulators of Ran's nucleotide state did not affect brood size (Table ). Thus, it is possible that Ran might have a function in oogenesis different from its GTPase activity, although it is also possible that RNAi fails to efficiently deplete Ran's regulators from the gonads.
Ran Is Required for Spindle Formation
Previous RNAi experiments have shown that Ran, RanGAP, and RanBP2 are involved in pronuclear and nuclear appearance and possibly also spindle formation (Gönczy et al., 2000
). IMA-2 has similarly been proposed to play a role in pronuclear formation (Zipperlen et al., 2001
). However, differential interference contrast (DIC) microscopy, the method of choice for those high-throughput screens, does not give detailed insight to the subcellular structures that are affected or the defects caused. We therefore retested these genes, as well as RCC1, IMB-1, and IMA-3, with a range of fluorescent markers and antibodies.
To investigate spindle formation in time-lapse microscopy we initially used a strain expressing a fusion protein of GFP and α-tubulin (Oegema et al., 2001
). In normal one-cell embryos the two centrosomes are associated with the sperm pronucleus until pronuclear meeting when the centrosomes border the junction between the two pronuclei (Figure A, −3:51. Note that in all time-lapse experiments time is indicated relative to anaphase onset; Video 1). The pronuclei then migrate together to the center of the embryo and the pronuclear envelopes break down as visualized by entry of soluble GFP::tubulin into the nuclear space (Figure A, −1:52). A spindle is rapidly formed, followed soon by anaphase (Figure A, 0:14), telophase, and cytokinesis (Figure A, :47). When Ran is depleted by RNAi (Figure C), several features are striking (4 of 5 embryos). First, the pronuclei are very small (Figure B, −5:29; Video 2) as reported previously (Gönczy et al., 2000
). Second, the embryos fail to set up a spindle, although the centrosomes are still capable of nucleating astral microtubules (Figure B, −1:10–1:59). Third, from being closely associated with the male pronucleus the centrosomes immediately move far apart and after several minutes undergo strong sideward movements, as in normal anaphase (Video 2). This process (“spindle rocking”) together with cleavage furrow ingression was used throughout this study to define anaphase in embryos where no spindle was formed. Fourth, in some embryos, the centrosomes are not properly positioned so that the first cleavage leads to an abnormal bisection of the embryo as seen in Figure B (11:40) where the posterior cell P1 is larger than the anterior AB. Taken together, these data indicate that Ran is required for spindle formation, but not for astral microtubule nucleation.
Figure 1 Ran is required for spindle formation. Still images from time-lapse microscopy are shown from wild-type (A) and Ran(RNAi) (B) embryos expressing GFP::α-tubulin. Anterior of the embryos is on the left and time (minutes:seconds) relative to anaphase (more ...)
Figure 4 Depletion of RCC1 leads to defects in chromosome segregation. Embryos expressing GFP::β-tubulin and GFP::histone H2B from either control worms (A) or worms depleted of RCC1 (B) were observed by time-lapse microscopy. Still images are shown with (more ...)
GTPase Activity of Ran Is Essential for Assembly of Spindle Microtubules
Chromosomes are proposed to play an active and important role in spindle formation (Compton, 2000
; Wittmann et al., 2001
). Depletion of Ran results in small pronuclei that are difficult to follow in DIC or GFP::α-tubulin recordings. The lack of a mitotic spindle in Ran(RNAi) embryos could therefore be a consequence of aberrant chromatin localization which in turn would cause a lack of chromatin-mediated microtubule-nucleating activity between the two centrosomes. To address this possibility, we created a transgenic strain that expresses GFP::histone H2B and GFP::β-tubulin by crossing the strains AZ212 (Praitis et al., 2001
) and WH204 (Strome et al., 2001
). This strain allowed us to precisely follow the localization and morphology of the chromatin concomitantly with centrosome position and microtubule dynamics. An example is shown in Figure A where three still pictures of a time-lapse recording represent prometaphase (−1:45), metaphase (−1:03), and anaphase (0:07) (Video 3).
Figure 2 Depletion of Ran, RanGAP, or RanBP2 causes similar defects on spindle formation. Embryos expressing GFP::β-tubulin and GFP::histone H2B from either control worms (A) or worms depleted of Ran (B), RanGAP (C), or RanBP2 (D) were observed by time-lapse (more ...)
When we targeted Ran, RanGAP, or RanBP2 with RNAi (Figure , E–F), joining of the two pronuclei was never observed, although oocyte pronuclear migration still took place (10 of 10 embryos where both pronuclei could be followed almost continuously; Videos 4–6). In approximately half of the cases (6 of 10) one of the pronuclei was eventually positioned in line between the two centrosomes, either in the middle (Figure B, closed triangle) or toward one of the centrosomes (Figure C, closed triangle). Most often (5 of 6) the pronucleus found between the centrosomes was from the oocyte (Figure , B–D, closed triangle), whereas the sperm pronucleus was more or less closely attached to one centrosome (Figure , B and D, open triangle). In other cases (4 of 10), neither of the pronuclei aligned between the centrosomes (our unpublished data). Importantly, spindles were never assembled in embryos where Ran, RanGAP, or RanBP2 was depleted, regardless of chromatin position. Analysis of fixed embryos was also carried out because the combination of live cell imaging and immunofluorescence provides resolution in time together with high sensitivity. Staining of microtubules with anti-α-tubulin antibodies confirmed our live cell recordings because we could not detect spindles in embryos depleted of Ran, RanGAP, or RanBP2, even in cases where mitotic chromatin was positioned between two centrosomes (Figure , A–C, F and G; 28 of 28 embryos). Immunofluorescence with antibody against phosphorylated histone H3 was used to confirm that the embryos were in prometaphase or metaphase. Thus, our data strongly indicate that the GTPase activity of Ran is essential for the assembly of microtubules that connect chromatin to the centrosomes.
Figure 3 RNAi embryos fail to assemble microtubules into spindles around mitotic chromatin. Embryos from GFP::histone H2B worms grown on either control bacteria (A and F), or bacteria expressing dsRNA corresponding to RanGAP (B), RanBP2 (C), ima-2 (D), imb-1 (E), (more ...)
RNAi of RCC1 Causes DNA Segregation Defects
Inhibition of RCC1 activity by the Ran mutant T24N has been shown to interfere with spindle microtubule assembly in Xenopus
egg extracts (Carazo-Salas et al., 1999
; Kalab et al., 1999
). Based on this and on our observation that Ran GTPase activity is necessary for spindle formation we predicted that inhibiting RCC1 expression would also block spindle formation by preventing formation of RanGTP. As with Ran depletions RCC1(RNAi) embryos are generally reduced in size (~15% shorter than wild type) and have smaller pronuclei (Figure ; our unpublished data). However, in contrast to the RNAi experiments described above the centrosomes in RCC1(RNAi) embryos stay associated with the sperm pronucleus. Instead of migrating to join the oocyte pronucleus at the normal position approximately one-third of the embryo length from the posterior pole the sperm pronucleus either remains at the cortex or moves only slightly away from it (14 of 14 embryos; our unpublished data). On joining, the two pronuclei migrate together toward the center of the embryo (Figure , −4:00; Video 7). Note that diffuse nucleoplasmic GFP::histone H2B staining is seen around the condensed chromatin before NE breakdown in wild-type embryos (Figure A, −3:12) but not in RNAi embryos (Figure B, −4:00; see Video 7 for earlier time points), suggestive of a defect in retention of GFP::histone H2B in the pronuclei. At this stage the centrosomes in the RCC1(RNAi) embryos moved away from the pronuclei (Figure , compare A and B at −1:48), creating a situation similar to Ran(RNAi) embryos in terms of distance between the centrosomes and the chromatin (Figure B). However, in the RCC1(RNAi) embryos this situation is rescued, giving rise to spindles that are indistinguishable from those in wild-type embryos (Figure , compare A and B at −0:12). Although the chromosomes are aligned between spindle microtubules in a seemingly normal metaphase (Figure B, −0:12) they clearly fail to segregate properly in anaphase (Figure B, 0:48). At later time points chromatin bridges are still observed across the cleavage furrow whereas wild-type embryos have completed cytokinesis and formed growing, spherical nuclei (Figure , A and B, 3:12). Consistent with the normal appearance of the mitotic spindle most (18 of 21) embryos divided into a larger AB and a smaller P1 daughter cell (Figure B).
Immunofluorescence analysis of RCC1(RNAi) embryos confirmed that DNA segregation is affected, yet mitotic spindles can assemble in embryos depleted of RCC1 (Figure H, compare missegregation of chromatin at the division of AB on the left with spindle formation in P1 on the right).
The observed pattern of an early spindle defect and later rescue could have been due to inefficient depletion of RCC1. To address this several approaches were taken: 1) In addition to RNAi-by-feeding we also tried to prevent RCC1 expression by injection of dsRNA into the gonads. 2) We targeted the RCC1 mRNA with three different dsRNAs covering in total the whole open reading frame. 3) Incubations on RNAi bacteria plates were prolonged up to 48 h. 4) RNAi was performed at either 20 or 25°C. None of these measures led to more severe effects on the spindle (our unpublished data). 5) Finally, we quantified the efficiency of RNAi by targeting RCC1 in a transgenic strain that expresses GFP::RCC1 in the germline. RCC1(RNAi) embryos from this strain had <5% GFP::RCC1 signal left when quantified by confocal microscopy (Figure C).
Centrosome Position Is Regulated by Ran System via Spindle Formation
To have another and more quantitative way of analyzing spindle formation, we measured the distance between the centrosomes relative to the total length of the embryo in time-lapse experiments. Aligning the data from nine different wild-type embryos relative to anaphase showed a very reproducible pattern (Figure D, Wild-type). Joining of the two pronuclei and setting up the spindle at NE breakdown do not change the distance between the centrosomes dramatically. Approximately 60 s before DNA segregation the spindle poles start to move apart, first slowly and later with higher velocity until maximum distance is reached and the embryo divides.
Although no spindle is formed in RanGAP(RNAi) embryos the centrosomes still rock from side to side, which was used as an anaphase indicator in aligning the curves from five independent embryos [Figure D, RanGAP(RNAi)]. The two centrosomes move rapidly apart ~3 min before mitosis to a final separation (Figure D) and position (Figure C) identical to normal embryos. Ran(RNAi) embryos followed a pattern very similar to RanGAP depletions (3 of 3 embryos). Due to the more dramatic movements of centrosomes and pronuclei seen when targeting Ran several frames in the time-lapse recordings were out of focus with respect to the centrosomes, making averaging more difficult. Instead, a representative example of a single Ran(RNAi) embryo is presented in Figure D, showing a similar pattern as RanGAP depletions. As described above, RCC1(RNAi) embryos also show premature separation of the centrosomes but are able to reverse this and build a functional spindle. When averaging 10 independent embryos the phenotype seems highly reproducible [Figure D, RCC1(RNAi)]. Initially, the centrosomes are associated with the sperm pronucleus but as microtubules connecting chromatin with the centrosomes appear, the centrosomes start to move prematurely apart to a maximum separation comparable with Ran(RNAi) and RanGAP(RNAi) embryos (Figure D, approximately −180 s; Figure B, −1:48). Later, the centrosomes move closer to each other again and reach an almost normal distance at metaphase (Figure D, approximately −30–0 s; Figure B, −0:18). During anaphase and telophase the centrosomes in RCC1(RNAi) embryos behave as in wild-type embryos (Figure D, 0–240 s), although chromatin segregation is clearly affected (Figure B, 0:48–3:12).
In summary, these data demonstrate that although spindle assembly is defective in the absence of Ran or RanGTP hydrolysis, the cues that control final centrosome position are functional. We suggest that the premature centrosome separation may be caused by a lack of Ran-dependent attachment of the centrosomes to the pronuclei.
IMA-2 and IMB-1, Homologues of Vertebrate Importin α and β, Are Essential for Spindle Formation
We initially analyzed IMA-2 and IMA-3 requirement by RNAi in embryos expressing GFP::histone H2B. ima-3(RNAi)
embryos were significantly reduced in size (our unpublished data) in agreement with a role of IMA-3 in oogenesis (Geles and Adam, 2001
; Table ). However, spindle formation seemed relative normal as judged from the presence of a metaphase plate and DIC images (our unpublished data) and IMA-3 was therefore not studied further. ima-2(RNAi)
embryos, on the other hand, had clear spindle defects (Figure D) and were examined more closely.
deletion allele was generated and isolated by the C. elegans
Gene Knockout Consortium. We obtained the ima-2(ok256)
allele and sequenced it to characterize the deletion. The ima-2(ok256)
allele lacks 1782 base pairs, from nt 325 downstream of the start codon to nt 283 downstream of the stop codon. This leads to a shortening of the protein from 532 to 76 amino acid residues from IMA-2 plus another 36 unrelated amino acid residues encoded by sequences downstream of the deletion. We therefore expect ima-2(ok256)
to be a null allele. In the wild-type genome the distance from the ima-2
stop codon to the start codon of the downstream gene F26B1.2 is only 647 base pairs. RNAi against F26B1.2 produced no detectable effects arguing against any role of F26B1.2 in our study (Fraser et al., 2000
By outcrossing the ima-2(ok256
) worms for several generations we obtained strain XA3513. Analysis of XA3513 by single worm PCR showed that ima-2(ok256
) homozygotes are viable but produce only inviable embryos. This demonstrates that IMA-2 is required either for proper oogenesis or early embryogenesis or both, consistent with its expression pattern (Geles and Adam, 2001
To study microtubule dynamics in the context of the ima-2(ok256)
allele we crossed XA3513 with WH204 (Strome et al., 2001
), giving rise to strain XA3503. Observation of embryos from ima-2(ok256
) worms revealed that the centrosomes initially stay relatively close to the sperm pronucleus and that pronuclear migration was normal (compare Figure B, first panel and Video 8 with Figure A and Video 1). However, the centrosomes then move apart to an intermediate degree of separation (Figure C, which displays the average of 5 embryos). The centrosomes remain separated, but no mitotic spindle was assembled and eventually the embryos enter mitosis as visualized by further centrosome separation, “spindle” rocking, and cytokinesis (Figure , B, 0:48–9:28, and C). In addition to the failure of spindle formation, the timing from pronuclear joining to rocking of the centrosomes was abnormally long in embryos from ima-2(ok256
) worms (504 ± 36 s [n = 5] compared with 332 ± 45 s in wild-type embryos [n = 3]). This suggests that IMA-2 could be involved in determining the onset or timing of mitosis.
Figure 5 Embryos devoid of IMA-2 or IMB-1 fail to form mitotic spindles. Embryos expressing GFP::β-tubulin from either a wild-type hermaphrodite (A) or from an ima-2(ok256) homozygous worm (B) were observed by time-lapse microscopy. Still images are shown (more ...)
Depletion of IMB-1 from embryos by RNAi give rise to a strong spindle defect like those seen in embryos from ima-2(ok256) worms and in Ran(RNAi) embryos. The centrosomes lose their association with the sperm pronucleus early (Figure D, −6:40; 3 of 3 embryos) and move far apart (Figure D, −3:20). The pronuclei never meet. The oocyte pronucleus partly aligns between the centrosomes but spindle assembly does not occur (Figure D, −3:20–0:40; see also Figure E and Video 15).
Disrupting the Ran Cycle Leads to Aneuploidy and Abnormal Chromatin Morphology
Following embryos that express GFP::histone H2B beyond the first division allowed us to investigate the fate of chromatin when the Ran cycle is abrogated. Measured from the completion of P0 division, AB in RNAi embryos divides at the same time as AB in control embryos. In contrast, P1 division in RNAi embryos is significantly delayed compared with wild-type embryos, resulting in prolonged three-cell embryo stages (Figure ; Videos 9–11; n > 50). Consistent with the finding that a functional Ran system as well as IMA-2 and IMB-1 are necessary for proper spindle function the DNA content of the cells is not equally distributed between the daughter cells and chromatin is often seen trapped at cell junctions (Figure , arrows). When any of the Ran-related genes was targeted by RNAi highly unstructured chromatin was present after the first division and throughout the next cell cycle (Figure ; our unpublished data; >90% for each gene), suggesting severe defects in NE reformation and perhaps DNA decondensation. In some cases, we observed that the chromatin eventually had time to round up in P1 as a consequence of the delayed division (Figure , E and F).
Figure 6 The Ran GTPase cycle, IMA-2, and IMB-1 are essential for normal chromatin appearance. Embryos from GFP::histone H2B worms grown on either control bacteria (A), or bacteria expressing dsRNA corresponding to Ran (B), RanGAP (C), RanBP2 (D), RCC1 (E), ima-2 (more ...)
NE Formation Is Dependent on RanGTP Production and Hydrolysis
Although RNAi against any component of the Ran cycle affected spindle formation, the embryos always went through several rounds of cytokinesis. This allowed us to investigate the role of the Ran GTPase cycle in NE formation after mitosis. Initially, we examined the distribution of nucleoporins by immunofluorescence with mAb414 (Davis and Blobel, 1987
). In wild-type embryos a bright and continuous mAb414 signal surrounds each nucleus (Lee et al., 2000
; Figure A). In contrast, when we disrupted the Ran system by targeting any of its components mAb414 stained aggregates, many of which were not associated with chromatin. In cases where association was detected the mAb414 signal was not uniform. We observed both situations within the same embryo for each of the genes analyzed (Ran, RanGAP, RanBP2, and RCC1, n > 10 for each gene). A single example for each gene is depicted in Figure , B–E. Similar results on depletion of Ran were reported by Bamba et al. (2002)
Figure 7 RNAi against Ran and its cofactors affects targeting of nuclear pore proteins. Embryos from GFP::histone H2B worms grown on either control bacteria (A), or bacteria expressing dsRNA corresponding to Ran (B), RanGAP (C), RanBP2 (D), RCC1 (E), ima-2 (F), (more ...)
Because NEs can assemble in the absence of nuclear pore complex insertion (Macaulay and Forbes, 1996
), we wished to determine whether the disrupted nucleoporin localization was a consequence of a lack of NE assembly. For this purpose, transgenic strains that express GFP fused to the inner nuclear membrane protein emerin (EMR-1; Lee et al., 2000
) were generated. GFP::EMR-1 can be used as marker for the NE. Embryos from these strains show a distinct NE signal as well as cytoplasmic staining like that seen with endoplasmic reticulum proteins (Figure A, Video 12). In older embryos the ratio of NE to total signal increases (our unpublished data), suggesting that early embryos may store EMR-1 protein in the endoplasmic reticulum for later use. Antibodies raised against EMR-1 give rise to similar staining (see below).
Figure 8 Disruption of the Ran GTPase cycle prevents nuclear envelope formation. Embryos from GFP::EMR-1 worms grown on either control bacteria (A), or bacteria expressing dsRNA corresponding to Ran (B and C), RanGAP (D), RanBP2 (E), RCC1 (F), ima-2 (G), or imb-1 (more ...)
In embryos depleted of Ran or any of its cofactors, RanBP2, RanGAP, or RCC1, no NE-like GFP::EMR-1 signal was detected after the first cell division in the majority of cases (67%) (Figure , B and D–F, Video 13). In other embryos, presumably where the RNAi had been less efficient, aberrant nuclei with a strong aggregation of GFP::EMR-1 next to the NE became apparent after a prolonged time (Figure C). Thus, the distribution of GFP::EMR-1 in RNAi embryos was similar to the mislocalization of nucleoporins described above. When endogenous EMR-1 was visualized by immunofluorescence with polyclonal antibodies (a gift of Y. Gruenbaum and K. Wilson), identical results were obtained (supplementary Figure 1).
As a further proof that a closed NE is not assembled after mitosis when the Ran cycle is inactivated, we determined whether soluble GFP::β-tubulin was uniformly distributed in the embryos. In normal embryos tubulin is excluded from the pronuclei and the nuclei and can therefore be used as marker for NE breakdown and reformation (Figure A and ). In contrast, Ran(RNAi) embryos never showed such exclusion after mitosis (Figure B; see also Figure , 4 of 4 embryos) and targeting the Ran cofactors also produced uniform GFP::β-tubulin staining (Figure , C and D, 5 of 6 embryos). We conclude that not only Ran but also its ability to cycle between RanGDP and RanGTP under the influence of RanGAP, RanBP2, and RCC1, is essential for an early step of NE assembly in vivo in these embryos.
Figure 9 Nuclear exclusion of soluble GFP::β-tubulin depends on the Ran GTPase cycle. Embryos from GFP::β-tubulin worms grown on either control bacteria (A), or bacteria expressing dsRNA corresponding to Ran (B), RCC1 (C), or RanGAP (D) were observed (more ...)
Depletion of IMA-2 or IMB-1 Affects NE Formation
We next wished to investigate whether IMA-2 and IMB-1 also play a role in regeneration of the NE after mitosis in vivo. Interestingly, multiple small structures surrounded by GFP::EMR-1 were detectable in ima-2(RNAi) embryos early (~1 min) after anaphase onset, when nuclei are formed in wild-type embryos (3 of 3 embryos, compare Videos 12 and 14). However, in ima-2(RNAi) embryos (Figure F) these structures grew only slowly in size and a significant amount of GFP::EMR-1 accumulated at the centrosomes (Figure , compare A and G). Presumably the appearance of multiple micronuclei after division is a consequence of the lack of spindle formation described above. The fact that the structures only grow slowly in size combined with the observation that ima-2(ok256) embryos do not show nuclear exclusion of soluble GFP::β-tubulin even late after mitosis (compare last panels in Figure , A and B, 4 of 4 embryos) suggests that IMA-2 is required either for fusion of nuclear membranes or for assembly of nuclear pore complexes.
RNAi against IMA-3 was clearly effective as judged from embryonic lethality (Table ) and alterations in embryo size and organization (our unpublished data). Despite this, nuclei were formed in ima-3(RNAi) embryos upon cell division and grew to nearly normal size (our unpublished data), illustrating that IMA-3 is not required for nuclear formation in early embryos. Embryos depleted of IMB-1 showed a strong defect in GFP::EMR-1 recruitment to the NE-like structures after mitosis (Figure H and Video16, 3 of 3 embryos). As with embryos where Ran or its cofactors had been targeted, imb-1(RNAi) embryos instead showed clustering of GFP::EMR-1 at the centrosomes. In addition, nuclear exclusion of soluble GFP::β-tubulin was not observed in embryos depleted of IMB-1 (our unpublished data; 3 of 3 embryos). Furthermore, immunofluorescence analysis demonstrated that chromatin in ima-2(RNAi) and imb-1(RNAi) embryos is most often not surrounded by a smooth and continuous nucleoporin staining (Figure , compare F and G with A; >80% for each gene).