Article |
Address correspondence to Erich A. Nigg, Dept. of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Tel.: 49-89-8578-3100. Fax: 49-89-8578-3102. E-mail: nigg{at}biochem.mpg.de
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Abstract |
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Key Words: CDC14; cytokinesis; central spindle; ZEN-4; AIR-2
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Introduction |
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In Schizosaccharomyces pombe, gene products apparently homologous to the components of the budding yeast MEN have originally been identified as regulators of septation, a process akin to cytokinesis (for review see Balasubramanian et al., 2000; Bardin and Amon, 2001). Hence, the corresponding regulatory pathway is referred to as the septum initiation network (SIN). Although many SIN genes are structurally similar to MEN genes, they appear to regulate primarily the deposition of the septum rather than the inactivation of Cdk1 (Cdc2p) (for review see Le Goff et al., 1999; McCollum and Gould, 2001). As a result, SIN mutants continue to segregate their DNA without undergoing cytokinesis. In a cytokinesis mutant, the SIN is also required to keep Cdk1 activity low and prevent entry into the next cell cycle. This latter function appears to be mediated by Clp1p/Flp1p, the S. pombe homologue of the Cdc14p phosphatase. Specifically, it appears that Clp1p/Flp1p may dephosphorylate both Cdc25p andWee1p, thereby maintaining Cdk1 in an inactive, tyrosine-phosphorylated state (Trautman et al., 2001). Fission yeast Clp1p/Flp1p also seems to be regulated by sequestration to the nucleolus during interphase. However, during mitosis, it has been observed on the SPBs, the spindle microtubules, and the cell division septum (Cueille et al., 2001; Trautman et al., 2001).
In metazoan organisms, the coordination of late mitotic events and cytokinesis with chromosome segregation remains poorly understood. Considering that metazoan cell division displays several features that are not encountered in yeast and vice-versa, the mechanisms regulating the exit from mitosis and the onset of cytokinesis in animal cells are expected to differ in important aspects from those emerging in yeast. For instance, considering that the nuclear envelope breaks down during mitosis in most multicellular eukaryotes, it is by no means clear that the nucleolar sequestration mechanism that controls Cdc14 activity in yeast is also operating in metazoan species.
In animal cells, the site of cleavage furrow formation is determined by the position of the mitotic spindle (Cao and Wang, 1996). During anaphase, a structure of antiparallel microtubule bundles, the so-called central spindle, forms between the separating chromosomes. This central spindle is essential for the completion of cytokinesis, as mutations in several of its components cause a failure in cytokinesis (Williams et al., 1995; Adams et al., 1998; Raich et al., 1998; Jantsch-Plunger et al., 2000). How the central spindle is assembled has not yet been elucidated in detail, but data from several organisms attribute an important role in the bundling of midzone microtubules to a mitotic kinesin-related motor protein, termed CHO1/Mklp1 in mammals (Sellitto and Kuriyama, 1988; Nislow et al., 1992), pavarotti in Drosophila (Adams et al., 1998), and ZEN-4 in C. elegans (Powers et al., 1998; Raich et al., 1998). Interestingly, the formation of the central spindle is prevented if Cdk1 activity persists, even though chromosome separation is not affected (Murray et al., 1996; Wheatley et al., 1997) Therefore, the reversal of the Cdk1-mediated inhibition of central spindle formation may constitute a key element in the coordination of cytokinesis with chromosome segregation.
Inspection of metazoan genomes reveals putative homologues for several components of the yeast MEN/SIN pathways, but for others, candidate homologues cannot readily be identified. Thus, it remains to be determined to what extent the yeast MEN/SIN circuits have been conserved during evolution. This question is particularly pertinent, as significant differences in the wiring of these circuits have been described even between budding yeast and fission yeast (for review see Balasubramanian et al., 2000; Bardin and Amon, 2001; McCollum and Gould, 2001). Of all the MEN/SIN gene products identified so far, the most highly conserved component is the phosphatase Cdc14p (Clp1p/Flp1p). In human cells, two CDC14 isoforms, CDC14A and CDC14B, have been described (Li et al., 1997, 2000). The function of these proteins remains poorly understood, although a recent study suggests a role for mammalian CDC14A in the centrosome cycle (Mailand et al., 2002). In support of this view, CDC14A localizes to centrosomes, whereas overexpressed CDC14B has been found at the nucleolus (Mailand et al., 2002). Furthermore, like its budding yeast counterpart, CDC14A can dephosphorylate Hct1/Cdh1p, at least in vitro (Bembenek and Yu, 2001).
To determine which, if any, of the identified yeast MEN/SIN components could play a role in mitotic exit and/or cytokinesis in metazoan cells, we studied the embryo of the nematode Caenorhabditis elegans, a widely used model for metazoan cell division. Specifically, we used RNA-mediated interference (RNAi) to deplete C. elegans worms of potential MEN/SIN homologues, and scored embryos for cell cyclerelated phenotypes. Of all candidate MEN/SIN components analyzed here, only the depletion of CeCDC-14, the C. elegans homologue of the S. cerevisiae Cdc14p phosphatase, produced embryonic lethality. Careful analysis of the corresponding phenotype revealed that CeCDC-14 is dispensable for progression of the nuclear cycles in the early embryo, but essential for central spindle formation and cytokinesis. This suggests that central spindle formation in animal cells depends critically on the dephosphorylation of one or several Cdk1-substrates.
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Results |
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To demonstrate that this ORF encodes a functional phosphatase, CeCDC-14 was expressed as a His-tagged fusion protein in bacteria. The purified protein was then used in phosphatase assays with the artificial substrate para-nitrophenyl phosphate (pNPP). 6HisCeCDC-14 hydrolyzed pNPP with a specific activity of 96 nmoles phosphate released min-1 mg-1, close to the specific activity reported for Cdc14p of S. cerevisiae (Taylor et al., 1997). This phosphatase activity was completely abolished upon mutation of the presumptive catalytic cysteine 295 to serine. Furthermore, dephosphorylation of pNPP by CeCDC-14 was efficiently inhibited by 200 µM sodium-orthovanadate (5% activity of control reaction) and only weakly inhibited by 20 mM sodium fluoride or 100 nM okadaic acid (72 and 87% activity of control reactions, respectively). A similar sensitivity to inhibitors had also been reported for Cdc14p (Taylor et al., 1997). Taken together, these data strongly indicate that the protein encoded by ORF C17G10.4c is the C. elegans homologue of S. cerevisiae Cdc14p. Thus, we hereafter refer to this protein as CeCDC-14.
To facilitate the study of CeCDC-14 in the embryonic cell cycles of C. elegans, a polyclonal antibody was raised against a COOH-terminal fragment of CeCDC-14 (spanning amino acids 352605). This fragment encompasses a region of the phosphatase domain that would be predicted to be conserved in all potential splice variants discussed above. Yet, in Western blots on total worm extracts, the affinity-purified antibody recognized a single band migrating at ca. 76 kD, very close to the size predicted for the smallest splice-variant (Fig. 1 A, lane 1). Furthermore, recombinant untagged CeCDC-14 comigrated exactly with the band detected in total worm extract (Fig. 1 A, lane 2), and both signals were completely eliminated upon addition of purified His-tagged CeCDC-14 (Fig. 1 A, lanes 3 and 4). Together, these results demonstrate the specificity of our antibody, and they confirm the identification of the endogenous protein as CeCDC-14.
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RNA-mediated interference with CeCDC-14 leads to failure of cytokinesis
The localization of CeCDC-14 to the central spindle and the midbody suggested an involvement of this protein in cytokinesis. To directly test this possibility, worms were injected with dsRNA corresponding to CeCDC-14. 24 h later, worms were dissected and the corresponding embryos analyzed. Most strikingly, all embryos derived from CeCDC-14 RNAi-treated worms were multinucleate, suggesting that they had undergone multiple cytokinesis failures. Identical results were obtained, regardless of the timing of dissection after worm injection (unpublished data; Table II). Analysis of CeCDC-14depleted embryos by immunofluorescence microscopy with antiCeCDC-14 antibodies (n = 25) did not show any staining, confirming the efficacy of protein elimination by RNAi (Fig. 2 A).
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In a minor proportion of embryos (6/36), the furrow only pinched in marginally before it regressed and cytokinesis failed. In some of these embryos, the pseudocleavage also appeared weaker or the spindle was placed more centrally than observed in wild-type embryos. Similar features have been observed in mutants defective in the establishment of polarity (Shelton et al., 1999; Rappleye et al., 2002) raising the possibility that the absence of CeCDC-14 might also have caused a polarity defect. However, when CeCDC-14depleted embryos were stained with antibodies against P granules, these were found to display the same posterior localization as in wild-type embryos (Fig. 2 C). Staining with antibodies against PAR-2 and PAR-3, two further markers of polarity, also failed to reveal aberrations in the establishment of polarity, and most CeCDC-14depleted embryos also displayed the characteristic flattening of the posterior spindle pole in anaphase (unpublished data; Keating and White, 1998). Thus, our results fail to support a major, direct role of CeCDC-14 in the establishment of polarity. On the other hand, considering that 15% of CeCDC-14depleted embryos did give indications of a polarity defect, it would be premature to rigorously exclude such a role. It remains possible that very low levels of residual phosphatase activity could suffice for a polarity-related function.
The central spindle fails to form in worms injected with CeCDC-14 dsRNA
Problems with cytokinesis could either reflect a failure to establish a stable central spindle (Jantsch-Plunger et al., 2000; Powers et al., 1998; Raich et al., 1998), or a failure of the actomyosin ring to assemble and contract correctly (Swan et al., 1998; Shelton et al., 1999). In order to inspect the state of the central spindle and the actin cytoskeleton in CeCDC-14depleted embryos, microtubules were stained with anti-tubulin antibodies and F-actin with phalloidin. In embryos injected with CeCDC-14dsRNA, bundled microtubules characteristic of the central spindle were absent, whereas the astral microtubules appeared normal (25 out of 25 embryos) (Fig. 3 A). Analysis of F-actin using FITC-phalloidin (Fig. 3 B) revealed the expected cortical actin staining in wild-type (n = 15), as well as in CeCDC-14 (n = 7) and ZEN-4depleted embryos (n = 9). The depleted embryos showed only partial actomyosin rings in telophase (Fig. 3 B), but this result needs to be interpreted with caution. As shown above (Fig. 2 B), the furrows in these depleted embryos regressed very rapidly, making it extremely difficult to capture the instant of maximal ingression for immunocytochemical analysis. Thus, we do not believe that either CeCDC-14 or ZEN-4 play a direct role in actomyosin ring formation, in agreement with previous reports on ZEN-4 (Powers et al., 1998; Severson et al., 2000). Instead, the regression of the contractile ring observed in CeCDC-14depleted embryos most likely stems from a failure of the central spindle to form correctly. Thus, our data identify CeCDC-14 as a novel regulator of central spindle formation in metazoan cells.
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Failure of ZEN-4GFP to localize to the spindle midzone in CeCDC-14RNAi embryos
From the analysis of fixed CeCDC-14depleted embryos, it was impossible to determine whether the mislocalization of ZEN-4 was due to an inability of ZEN-4 to localize to the central spindle, or alternatively, reflected an unstable association of ZEN-4 with the spindle midzone. The latter mechanism had previously been described to underlie the ZEN-4 mislocalization phenotype in embryos injected with dsRNA for the C. elegans INCENP homologue, ICP-1 (Kaitna et al., 2000). To distinguish between the two possibilities, worms carrying a functional ZEN-4GFP fusion (Kaitna et al., 2000) were injected with CeCDC-14 dsRNA, and CeCDC-14depleted and control embryos were then observed in real time. In wild-type embryos, ZEN-4GFP began to localize to the central spindle in anaphase, and subsequently formed a bright spot at the position of the midbody in telophase (n = 4) (Fig. 5, left). However, in embryos depleted of CeCDC-14, no transient accumulation of ZEN-4GFP could be observed at any time point (n = 6) (Fig. 5, right two panels). From the comparison of these results with those reported for embryos depleted of ICP-1 (Kaitna et al., 2000), it seems clear that depletion of CeCDC-14 disturbs the organization of the central spindle more completely than depletion of ICP-1.
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Discussion |
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Depletion of CeCDC-14 but not other MEN/SIN homologues causes embryonic lethality
Surprisingly, our RNAi-based survey of potential C. elegans MEN/SIN homologues revealed an embryonic requirement only for the CeCDC-14 phosphatase. Several factors may contribute to explain this finding. First, two of the MEN/SIN components analyzed (Dbf2p and Mob1p) have more than one potential homologue in the C. elegans genome, raising the possibility of redundancy. In these cases, it might be necessary to simultaneously deplete all potential homologues in order to observe a phenotype. Second, it is possible that some of the MEN/SIN components serve to fulfill requirements specific to yeast cell budding or fission, respectively, and these functions may not have been conserved during evolution of metazoan organisms. Third, whether or not a particular gene product is required for cell cycle progression may depend on the developmental context. Consistent with this possibility, we have observed that depletion of two C. elegans kinases structurally related to MEN/SIN components (Dbf2p of S. cerevisiae and Sid1p of S. pombe) produced an F1 arrest at the L1 stage and degeneration of the germ line, respectively.
CeCDC-14 regulates cytokinesis in C. elegans embryos
Our studies provide strong evidence for a key role of the metazoan Cdc14 phosphatase in cytokinesis. As a result of this critical requirement, C. elegans embryos depleted of CeCDC-14 display early embryonic lethality. The phenotype of embryos derived from CeCDC-14dsRNA-injected worms indicates that CeCDC-14 is important for the regulation of cytokinesis per se, independent of a possible role in cell cycle control. Previous studies on the budding yeast Cdc14p protein had not revealed a direct role for this phosphatase in cytokinesis. However, because cdc14-ts mutants arrest at late anaphase, any requirement for Cdc14p at later stages of the cell cycle might have escaped detection. In the case of fission yeast, some cytokinesis defects could be observed in the absence of Clp1p/Flp1p, particularly in conjunction with mutations of other SIN components (Cueille et al., 2001). Thus, this phosphatase may play a role in the regulation of cytokinesis not only in animal cells but also in yeast.
CeCDC-14 depletion does not affect the embryonic C. elegans cell cycle
In both S. cerevisiae and S. pombe, Cdc14p and Clp1p/Flp1p have been implicated in the regulation of Cdk1 activity, albeit at different stages of the cell cycle. In striking contrast, in C. elegans embryos depleted of CeCDC-14, the nuclear cycles continued with similar timing as in wild-type embryos. Taken at face value, this result indicates that the C. elegans Cdc14 phosphatase is required for cytokinesis but not for other aspects of cell cycle control. Of course, it is difficult to rigorously exclude that some residual CeCDC-14 protein, at levels undetectable by immunofluorescence microscopy, might be sufficient to fulfill a potential cell cycle function. In an attempt to maximize the depletion of CeCDC-14, we have also analyzed embryos that were dissected after prolonged incubation of injected worms. This has been reported to enhance depletion of some proteins (Rappleye et al., 2002), but in our experiments did not give rise to additional phenotypes. The vast majority of treated embryos was defective in both polar body extrusion and cytokinesis, and yet progressed through the cell cycle with near-normal kinetics. At the very least, this suggests that embryonic cell cycle progression is much less dependent on Cdc14 activity than cytokinesis.
It is important to bear in mind that embryonic cell cycles may lack certain controls that are operating at later developmental stages. For instances, embryonic cycles appear to be devoid of checkpoint mechanisms, as indicated by their inability to arrest in response to nocodazole (Hyman and White, 1987; Kitagawa and Rose, 1999). Thus, although the direct regulation of cytokinesis may well be the only essential function of CeCDC-14 in the embryonic cycles, additional functions may be required at larval stages. We cannot presently test this possibility, because the CeCDC-14depleted embryos die before hatching. Thus, to further elucidate a potential role for CeCDC-14 in the regulation of somatic cell cycles, temperature-sensitive mutants will be required.
Depletion of CeCDC-14 interferes with central spindle assembly
In embryos depleted of CeCDC-14, bundled midzone microtubules did not assemble, and the kinesin-related motor ZEN-4 failed to localize to the spindle midzone. This behavior of ZEN-4 is different from that observed in embryos depleted for the INCENP protein ICP-1, in which ZEN-4 could locate to the central spindle, albeit transiently (Kaitna et al., 2000). The phenotype observed here could be explained in two ways: first, it is possible that CeCDC-14 regulates an upstream factor essential for central spindle formation. In this scenario, the failure of ZEN-4 to localize to the central spindle in CeCDC-14depleted embryos would simply reflect the absence of stable midzone microtubules. Alternatively, ZEN-4, and/or its partner CYK-4, could themselves be regulated by CeCDC-14. Specifically, one could argue that ZEN-4 and/or CYK-4 need to be dephosphorylated in order to localize to the forming central spindle, and that in their absence, microtubule bundles might disassemble. To test this second possibility, we have phosphorylated recombinant ZEN-4/CYK-4 complexes by Cdk1-cyclin B in vitro, and then used these phosphoproteins as substrates for CeCDC-14. However, although the recombinant phosphatase readily dephosphorylated histone H1, no dephosphorylation of either ZEN-4 or CYK-4 could be observed (M. Mishima, U. Gruneberg, and M. Glotzer, personal communication). In future studies, it will thus be interesting to consider additional candidate substrates of CeCDC-14. These might include microtubule-stabilizing factors or other regulatory proteins involved in the process of central spindle formation.
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Materials and methods |
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For the depletion of potential C. elegans MEN/SIN homologues other than CeCDC-14, the following ESTs were used for preparing dsRNA or feeding constructs, as described above: yk157e4, yk249f3 (R11G1.4), yk467f11 (T12B3.4), yk8f12 (T19A5.2), yk50a8 (T20F10.1), yk462g7, and yk373c2 (F09A5.4). F38H4.10 and C33F10.2 were amplified from C. elegans cDNA and TA cloned either into L440 for feeding experiments or into pCRII-TOPO (Invitrogen). For in vitro transcription, the inserts were amplified with M13-FW and RV-primers and transcribed using T7 and SP6 polymerase. All ESTs used in this study were provided by Dr. Yuji Kohara (National Institute of Genetics, Mishima, Japan).
DIC filming of worm embryos
All filming of embryos was conducted at 20°C. Worms were dissected in a drop of PBS on an 18 x 18-mm coverslip and mounted onto a 2% agar pad. The coverslip was sealed with silicon glue and embryos were recorded with a 63x/1.4 objective using DIC optics on a Zeiss Axiovert S 100 microscope equipped with a JAI CV-M50 camera and Image-Pro Express 4.0 software. Single frames were then exported into Adobe Photoshop.
Time-lapse recording of ZEN-4GFP and histone H1-GFP embryos
For real-time recordings of ZEN-4GFP in C. elegans embryos, strain MG170 (zen-4 [or 153ts]); (xsEx6 [zen-4GFP]]) (Kaitna et al., 2000) was used. This strain was propagated at 25°C. Untreated or RNAi-injected MG170 worms were dissected as described above, and time-lapse recordings of GFP fluorescence were carried out as described previously (Jantsch-Plunger et al., 2000), using a Zeiss Axioplan 2 microscope with a piezo stepper (Physik Instrumente), a CoolSnap camera for image acquisition and Metamorph software. The exposure time for GFP fluorescence was 100 ms, and six frames, 2 µm apart, were taken every 15 s. Time-lapse recordings of histone H2B-GFP embryos were performed in a similar manner, with the exception that only two frames, 1.5 µm apart, were taken every 6 s in order to increase temporal resolution. Filming of histone-GFP embryos was carried out at 25°C. For data analysis, each stack of GFP images was projected onto a single plane, and the projections were processed for figures using Adobe Photoshop.
Phosphatase assays
Full-length CeCDC-14 was cloned into pQE32 (QIAGEN) and purified according to standard procedures using Ni-NTA agarose (QIAGEN). The protein was eluted with 20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 200 mM imidazole, and the imidazole subsequently removed by passing the eluate over a 5-ml HiTrap desalting column (Amersham Biosciences). Purified CeCDC-14 was stored at 80° in 25 mM Hepes, pH 7.3, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol. Phosphatase activity was measured in reaction buffer consisting of 50 mM imidazole, pH 6.9, 1 mM DTT, 1 mM EDTA as described (Taylor et al., 1997), using 1 mg/ml para-nitrophenyl phosphate (Sigma-Aldrich) and 2006,400 ng of enzyme. Control reactions were carried out using the phosphatase-dead CeCDC-14 (cysteine 295 mutated to serine).
Antibodies
For the production of CeCDC-14-specific antibodies, rabbits were immunized with a GSTCeCDC-14 aa 352605 fusion protein expressed in E. coli. Antibodies were affinity-purified using a 6His-CeCDC-14 fusion protein coupled to Affi-Gel 10 and used at a final concentration of 0.25 µg/ml.
The antibodies specific for ZEN-4 and AIR-2 have been described (Jantsch-Plunger et al., 2000; Kaitna et al., 2002). Tubulin was stained with the mouse monoclonal antibody DM1A (Sigm-Aldrich). Antibodies recognizing phospho-histone H3 (serine10) were obtained from Upstate Biotechnology. Affinity-purified sheepanti-GFP antibody was a gift from Dr. Francis Barr (Max-Planck Institute for Biochemistry). AntiPGL-1 antibodies and antibodies against PAR-2 and PAR-3 were provided by Drs. Susan Strome (Indiana University, Bloomington, IN) and Ken Kemphues (Cornell University, Ithaca, NY), respectively.
Insect cell expression
For baculovirus expression, full-length C17G10.4c was amplified by RT-PCR from total worm cDNA using an Advantage RT-for-PCR kit (CLONTECH Laboratories, Inc.), TA-cloned into pCRII-TOPO (Invitrogen) and subcloned into the baculovirus transfer vector pAcSG2 (Becton-Dickinson). Recombinant baculoviruses were generated by cotransfecting Sf9 cells with the C17G10.4c transfer vector and BaculoGold-DNA (Becton-Dickinson) according to the manufacturer's protocols.
Immunoblotting
Total worm lysate was prepared by resuspending a worm pellet of 100 µl in 200 µl urea-buffer (Knop et al., 1996). The lysate was heated at 65°C for 10 min and 5 µl were applied per lane of an SDS-PAGE minigel. The separated proteins were transferred to Protran membrane (Schleicher and Schuell) and probed with affinity-purified antiCeCDC-14 antibodies at a concentration of 0.25 µg/ml. For analysis of untagged recombinant CeCDC-14 protein, baculovirus-infected Sf9 cells were harvested 48 h postinfection with the C17G10.4c-virus. The cell pellet was lysed at 4 x 106 cells/ml in 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% IGEPAL, 1 mM Pefabloc (Roche), and a protease inhibitor cocktail (Roche). The lysate was centrifuged for 10 min at 14,000 rpm in a table top centrifuge and the supernatant diluted 1:10 with PBS. 5 µl was loaded per lane of an SDS-PAGE minigel and processed as described above.
Immunofluorescence microscopy
For staining of phospho-histone H3, ZEN-4, AIR-2, and CeCDC-14, embryos were freeze cracked and fixed in 20°C methanol for 20 min as described (Jantsch-Plunger et al., 2000). Phalloidin staining was performed using a modified version of the published protocol (Strome, 1986). The fixation/staining solution was 4% paraformaldehyde, 50 mM Pipes, pH 6.8, 5 mM EGTA, 300 nM FITC-phalloidin. For P granule staining embryos were fixed for 10 min in cold methanol followed by 10 min in cold acetone ass described (Kawasaki et al., 1998). Stacks of immunofluorescence images 0.2 µm apart were collected using a Deltavision microscope (Applied Precision) and a 100x/1.4 oil objective. The three-dimensional stacks (typically 3545 planes) were computationally deconvolved, projected onto a single plane and further processed for figures in Adobe Photoshop.
Online supplemental material
Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200202054/DC1) documents the phenotypes observed upon depletion of the putative Dbf2p homologue T20F10.1 and the putative Sid1p homologue T19A5.2.
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Footnotes |
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* Abbreviations used in this paper: APC/C, anaphase promoting complex/cyclosome; DIC, differential interference contrast; MEN, mitotic exit network; pNPP, para-nitrophenyl phosphate; RNAi, RNA-mediated interference; SIN, septum initiation network; SPB, spindle pole body.
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Acknowledgments |
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U. Gruneberg is the recipient of an EMBO long-term postdoctoral fellowship and was initially supported by Deutsche Forschungsgemeinshaft grant 703/1 to Dr. Anton Gartner.
Submitted: 12 February 2002
Revised: 11 July 2002
Accepted: 25 July 2002
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