Correspondence to Anjon Audhya: aaudhya{at}ucsd.edu; or Karen Oegema: koegema{at}ucsd.edu
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Introduction |
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The embryo of the nematode Caenorhabditis elegans recently emerged as a powerful system for studying cell division. In C. elegans, RNA-mediated interference (RNAi) can be used to generate oocytes that are depleted of targeted essential proteins in a process that does not depend on intrinsic protein turnover (Oegema and Hyman, 2005). The first mitotic division of the depleted embryos can be monitored after fertilization. Several genome-wide RNAi-based screens have identified the set of nonredundant genes that is required for embryonic viability (Gunsalus and Piano, 2005). Additional screens in which embryos that were depleted of each of these gene products were imaged by differential interference contrast (DIC) microscopy defined further the subset of these genes that have essential roles in cell division (Gönczy et al., 2000; Piano et al., 2000; Zipperlen et al., 2001; Sönnichsen et al., 2005). This approach successfully identified most proteins that were known to function during cytokinesis. A small number of new gene products also were identified, including one that corresponds to an uncharacterized, potential RNA-binding protein (Y18D10a.17) that we named CAR-1 (see Results section).
Sequence analysis showed that CAR-1 is a member of the Scd6 family of proteins. Scd6 family members contain several distinct sequence features, including regions of low complexity that are enriched in charged residues (e.g., RS and RG motifs) that are typical of RNA-binding proteins (Dreyfuss et al., 1993), and a recently defined FDF domain of unknown function (Anantharaman and Aravind, 2004). All Scd6 family members also have a divergent Sm domain at their NH2-termini (Anantharaman and Aravind, 2004). Sm and Sm-like domains are present in a variety of proteins that have been implicated in RNA metabolism, including small nuclear ribonucleoprotein particles that contribute to pre-mRNA splicing, RNA processing, and telomere replication (for review see Kambach et. al., 1999), and protein complexes that are implicated in mRNA decapping and degradation (for review see Coller and Parker, 2004). Sm domains typically associate with other Sm domains to form heptameric or hexameric toroids that bind to short uracil-rich stretches of RNA (Toro et al., 2001; Thore et al., 2003). However, the sequence divergence within the atypical Sm domain that is found in Scd6 family members is predicted to confer unique RNA binding properties to this protein family (Anantharaman and Aravind, 2004).
Because the sequence features of the conserved Scd6 protein family were recognized only recently, their function remains unclear. To better understand the function of Scd6 family proteins, and how depletion of a predicted RNA-binding protein results in a cytokinesis defect, we characterized the C. elegans Scd6 homologue, CAR-1. We show that CAR-1 is a component of a multiprotein complex that also contains the DEAD box RNA helicase, CGH-1, and a Y-boxcontaining protein, CEY-2. CAR-1 and CGH-1 localize to RNA-containing P-granules that concentrate in the germline precursors, and to smaller cytoplasmic particles that are present in the gonad and in all cells of early embryos. Depletion of CAR-1 results in a specific defect in the microtubule cytoskeleton that becomes pronounced after anaphase onset, when assembly of interzonal microtubule bundles is impaired severely and cytokinesis fails. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of RNAs that is required for anaphase spindle structure and cytokinesis during early embryogenesis.
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Results |
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To determine if CAR-1 function also is required at other stages of development, we analyzed a deletion allele of car-1. The deletion tm1753, which became available from the Japanese National Bioresource Project during the course of this work, is predicted to delete the COOH-terminal 225 amino acids of CAR-1, including the RGG box and the FDF domain. Homozygous car-1(tm1753) mutant embryos laid by heterozygous mothers contain maternally loaded CAR-1 that allows them to progress through the early stages of embryogenesis normally. Surprisingly, homozygous car-1(tm1753) mutant embryos hatched and developed through the larval stages to form apparently normal adult hermaphrodites. However, homozygous adult car-1(tm1753) hermaphrodites laid significantly fewer embryos than did the wild type (22 ± 6 in 24 h at 20°C vs. 48 ± 5 for wild-type; n = 5), and all of these embryos failed to hatch. Embryos laid by homozygous car-1 mutant mothers exhibited a cytokinesis defect identical to that observed in embryos that were depleted of CAR-1 by RNAi (Fig. 1 C; Video 1; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200506124/DC1). These results suggest that our RNAi phenotype corresponds to a complete loss of maternally supplied CAR-1. In addition, we conclude that although zygotic transcription of car-1 is not required for development to adulthood, maternally loaded CAR-1 is essential for cytokinesis during early embryogenesis.
CAR-1 localizes to RNA-containing particles
To determine how depletion of CAR-1 leads to a defect in embryonic cytokinesis, we examined its localization. An affinity-purified antibody to the COOH terminus of CAR-1 (Fig. 1 A) detected two closely spaced bands on Western blots that were reduced by >95% by RNAi of car-1 (see Fig. 3 D). CAR-1 localized to cytoplasmic particles whose size and distribution varied during the early embryonic divisions (Fig. 2 A). Particles were not detected in car-1(RNAi) embryos (Fig. 2 B), which confirmed the specificity of the localization. From the latter half of the first division onward, CAR-1 localized prominently to large particles that were similar in size and distribution to P-granules, which are enriched in the germline precursors and contain poly(A)+ RNAs and several proteins that are predicted to bind RNA (Strome and Wood, 1982; Seydoux and Fire, 1994). By performing immunofluorescence in a strain expressing GFP:PGL-1 (Cheeks et al., 2004), we confirmed that a subset of CAR-1 colocalizes with PGL-1 to P-granules (Fig. 2). Depletion of CAR-1 did not affect P-granule formation or distribution (Fig. 2 B), which indicated that CAR-1 is not necessary for either of these events.
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To examine CAR-1 particle dynamics directly, we filmed embryos expressing a functional GFPLAP:CAR-1 fusion (see Fig. 3 B, D, and E and Video 4). The formation of the small GFPLAP:CAR-1containing particles in both daughter cells after completion of the first cytokinesis was particularly prominent in these sequences. The number of small particles increased as embryos proceeded into the four- and eight-cell stages, but the particles were not detected in older embryos. In summary, consistent with the RNA-binding motifs in its primary sequence, CAR-1 localizes to P-granules that are partitioned to the germline precursors and to smaller particles that are present in all cells of early embryos.
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Surprisingly, after depletion of endogenous CAR-1, GFPLAP: CAR-1N localized identically to full-length GFPLAP:CAR-1 (Fig. 3 C). This result indicates that the CAR-1 Sm domain is dispensable for its localization to P-granules and smaller particles. Together with the fact that PGL-1 targets normally in CAR-1depleted embryos (Fig. 2 B), this result indicates that inhibition of CAR-1 function does not prevent assembly of P-granules or the smaller particles that are present in all cells.
CAR-1 associates with the essential RNA helicase CGH-1 and the Y-boxcontaining protein CEY-2
The above results suggested that CAR-1 interacts with other RNA-binding proteins to execute its function in cytokinesis. To identify such proteins, we used a tandem affinity purification scheme (Cheeseman et al., 2004). Protein complexes containing GFPLAP:CAR-1 were purified by immunoprecipitation with antibodies to GFP, released by cleavage with the tobacco etch virus protease, and reisolated by binding to S-protein agarose. Proteins eluted from the S-protein agarose with urea were analyzed by solution mass spectrometry. Under these stringent purification conditions, we obtained significant sequence coverage for CAR-1 and two additional proteins, the RNA helicase, CGH-1, and the Y-box domaincontaining protein, CEY-2, which also is predicted to bind RNA (Fig. 4, A and B).
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To determine if the interaction between CAR-1 and CGH-1 requires RNA, we used antibodies to immunoprecipitate CAR-1 from extracts after incubation in the presence or absence of RNaseA. Whereas CGH-1 was detected in CAR-1 immunoprecipitates from control extracts, no CGH-1 was present after RNase treatment (Fig. 4 C). We conclude that CAR-1 is a component of an RNase-sensitive, multiprotein complex of conserved RNA-binding proteins.
CGH-1 controls the localization of CAR-1
To explore the functional relationship between CAR-1 and CGH-1, we determined the effect of depleting each protein on the localization of the other. Because depletion of CGH-1 results in sterility, we examined CAR-1 localization in the syncytial gonad of depleted worms. The gonad is composed of a cylindrical shell of meiotic nuclei that surrounds a common cytoplasmic core called the rachis. CAR-1 weakly colocalized with PGL-1 to P-granules, which surround the nuclei in the central portion of the gonad. However, most CAR-1 was present in smaller granulesdistributed throughout the cytoplasm of the rachisthat did not contain PGL-1 (Fig. 4, D and E).
In CGH-1depleted worms, the localization of CAR-1 in the gonad was perturbed dramatically. CAR-1 still weakly localized to P-granules around the nuclei, but the small CAR-1containing particles were absent. Instead, CAR-1 accumulated in large, bar-shaped structures in the center of the rachis that appeared to form sheets (Fig. 4 E). This result was confirmed using a strain harboring a cgh-1 deletion allele (ok492; not depicted); an identical reorganization was observed in living CGH-1depleted worms expressing GFP:CAR-1 (Fig. 4 F). The localization of GFP:PGL-1 also was altered after depletion of CGH-1. Like CAR-1, GFP:PGL-1 was still detected in P-granules, but it also was associated prominently with the aberrant CAR-1containing structures in the rachis. In contrast to depletion of CGH-1, inhibition of CAR-1 did not affect the targeting of CGH-1 or PGL-1 to either type of granule. Similarly, depletion of CEY-2 or PGL-1 did not affect the targeting of CAR-1 or CGH-1. These results suggest that CGH-1 controls the formation of the small CAR-1containing particles, whereas CAR-1 is not required for the formation of either particle type (Table I; see also Figs. 2 B and 3 C).
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CAR-1depleted embryos exhibit a pronounced defect in anaphase spindle structure
To analyze spindle structure directly, we examined embryos expressing GFP:-tubulin or coexpressing GFP-histone H2B and GFP:
-tubulin (Fig. 6 A; Video 7). Although spindle length before anaphase was not affected appreciably by CAR-1 depletion, subtle defects in spindle structure during mitosis and meiosis were evident before the onset of chromosome segregation (Fig. S3). Consistent with this, lagging chromosomes and chromosome bridges were observed frequently in CAR-1depleted embryos (n = 45/50) (Fig. 6 B, 20/40-s panels; Video 8). At anaphase onset, a dramatic defect was apparent in CAR-1depleted embryos; the spindle poles separated abruptly and prematurely, and the interzonal microtubule bundles that normally form between the separating chromosome masses were not detectable (Fig. 6 A, arrows in 100-s panel). Average plots of spindle pole separation versus time confirmed this reproducible anaphase "spindle snapping" defect (Fig. 6 C). These results indicate that CAR-1 is required for assembly of the anaphase spindle; this may explain the nature of the cytokinesis defect that is observed in CAR-1depleted embryos.
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Partial depletion of CGH-1 phenocopies CAR-1 inhibition
CAR-1 associates with the helicase, CGH-1, whose inhibition results in penetrant sterility. To address whether CGH-1 function is required for embryonic cytokinesis, we analyzed partial CGH-1 depletions. 24 h after injection of a dsRNA targeting cgh-1, all worms ceased embryo production. However, 2224 h after injection, several worms were able to fertilize up to two oocytes. The resulting embryos were osmotically sensitive, but could be imaged in utero or by using specialized media to provide osmotic support (see Materials and methods). Strikingly, the defects observed in partial CGH-1depleted embryos were almost identical to those in CAR-1depleted embryos (Fig. 8). Cytokinesis failed in most partial CGH-1depleted embryos (n = 11/19), and imaging of the microtubule cytoskeleton confirmed the absence of interzonal microtubule bundles (Fig. 8 B). In addition, AIR-2 accumulated on mitotic chromosomes but failed to target to interzonal microtubules (Fig. 8 C). Consistent with the analysis in gonads (Fig. 4, D and E), localization of CAR-1 was perturbed severely in partial cgh-1(RNAi) embryos. Most of the CAR-1 accumulated in small, bar-like structures that did not migrate to the posterior during the first mitotic division (Video 9).
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General inhibition of translation does not phenocopy CAR-1 and partial CGH-1 depletions
CGH-1 has been proposed to function in the regulation of mRNA translation (Navarro et al., 2001). To determine whether the observed cytokinesis defect in CAR-1 and partial CGH-1 depletions could arise from a general defect in the translation of mRNA in the gonad and early embryo, we examined whether loss of interzonal microtubules was a consequence of inhibiting translation. Depletions of six different ribosomal subunits (RPS-1, -3, -5, -11, -18, and -23) each resulted in penetrant sterility (unpublished data). Consequently, we performed partial depletions of each ribosomal subunit, and examined the localization of GFP:AIR-2 in the final few embryos produced before onset of sterility. In every case, GFP:AIR-2 accumulated on interzonal microtubule bundles (Fig. S4 and not depicted). Defects in cytokinesis were not observed after partial depletion of ribosomal subunits, although embryos were osmotically sensitive and exhibited other pleiotropic phenotypes, consistent with those previously reported for general inhibition of translation (Gönczy et al., 2000). These results suggest that the cytokinesis defect after CAR-1 or partial CGH-1 depletion is not a consequence of generally inhibiting translation in the gonad and during early embryogenesis.
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Discussion |
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CAR-1 forms a conserved complex with a DEAD box RNA helicase and a Y-box protein
CAR-1 copurifies with the essential DEAD box RNA helicase, CGH-1, and the Y-box protein, CEY-2. In Drosophila, a maternally expressed protein complex containing the CGH-1 homologue, Me31B, and a Y-box protein was described (Wilhelm et al., 2000; Nakamura et al., 2001). The CAR-1 homologue in Drosophila, trailerhitch, is a component of this complex (Wilhelm, J., personal communication). The amphibian homologues of CGH-1 and CAR-1, Xp54 and RAP55, also were enriched in mRNP particles that were isolated from oocytes (Ladomery et al., 1997; Lieb et al., 1998). This suggests that the regulation of maternally supplied RNA, by a complex containing a Scd6 family protein and a DEAD box helicase, during oogenesis and early embryogenesis is conserved among metazoans.
Partial depletion of CGH-1 phenocopies depletion of CAR-1, which supports the idea that these two proteins function together. However, the CGH-1 depletion and deletion phenotypes are more severe than that of CAR-1. Adults that are homozygous for a deletion in car-1 lay embryos that fail to complete cytokinesis, whereas adults that are homozygous for a deletion in cgh-1 are sterile. Consistent with the difference in phenotypic severity, depletion of CAR-1 does not disrupt the localization of CGH-1, PGL-1, or a nonfunctional CAR-1 truncation lacking the Sm domain. These data indicate that although CAR-1 may function in the context of P-granules or smaller cytoplasmic particles, it is not required for their formation. In contrast, depletion of CGH-1 results in the loss of the small CAR-1containing particles, and causes CAR-1 and PGL-1 to accumulate in aberrant sheet-like structures in gonad. The discrepancy between our data, and a previous study that reported that PGL-1 targeting was normal after RNAi of cgh-1 (Navarro et al., 2001), probably is due to the more severe nature of our perturbation. The cgh-1(RNAi) worms that were examined previously were the final surviving F1 progeny of hermaphrodites that were injected with dsRNA against cgh-1. In contrast, we analyzed the gonads of hermaphrodites that were injected as L4 larvae.
CAR-1/CGH-1: a link between maternal RNA regulation and anaphase spindle structure
In Xenopus extracts, RNPs containing the mRNA export factor, Rae1, concentrate around chromatin and in the centers of the centrosomal microtubule asters, where they have been proposed to have a direct, translation-independent role in spindle assembly (Blower et al., 2005). Although, we cannot rule out a direct role for RNAs regulated by CAR-1/CGH-1 in spindle structure, the fact that CAR-1/CGH-1containing particles do not concentrate in the vicinity of the spindle makes this unlikely. Instead, we favor the idea that CAR-1 and CGH-1 modulate spindle dynamics indirectly by regulating the translation of a specific set of maternally supplied mRNAs in the gonad and early embryo.
Why is the function of CAR-1/CGH-1 restricted to the gonad and early embryo? The C. elegans gonad is a syncytium, lined by 800 nuclei, that progresses in an orderly fashion from a distal mitotic zone, through the various stages of meiotic prophase, and ultimately become packaged in oocytes. During this process, the meiotic nuclei also serve a nurse function, and supply the common cytoplasm with mRNA (Gumienny et al., 1999). Oocytes containing maternally supplied protein and mRNA bud off the tip of the gonad and are fertilized to form embryos. We speculate that spatial and temporal control of translation is important to maintain the local environments in the different regions of the gonad and early embryo. Regulation of maternally supplied RNA would be critical until the embryonic cells begin to transcribe their own genomeapproximately the time that CAR-1/CGH-1containing particles can no longer be detected in embryos. Because the dynamics of cytoskeletal polymers are very sensitive to the concentration of their regulators, it seems possible that the anaphase spindle defect in CAR-1depleted embryos results from the translational misregulation of a component of the microtubule cytoskeleton.
A role in translational competence would be similar to that proposed previously for the RNA binding protein, CPEB, the inhibition of which results in defects in spindle structure in Xenopus oocytes (Groisman et al., 2000). Homologues of CPEB regulate the translational competence of mRNAs in oocytes and neurons by promoting the polyadenylation and activation of mRNAs that are stored in a deadenylated, translationally silenced state (de Moor et al., 2005). A possible functional connection with CAR-1 is suggested by the fact that clam CPEB/p82 has been copurified with the CGH-1 homologue, p47 (Minshall et al., 2001). However, because we did not isolate any of the C. elegans homologues of CPEB in our CAR-1 purification, and inhibition of cpb-3, the closest C. elegans CPEB homologue, does not result in embryonic lethality (Luitjens et al., 2000), additional experiments are needed to test this hypothesis.
A role for CAR-1 and its associated helicase, CGH-1, in regulating the translational competence of maternal RNAs in the gonad and early embryo is consistent with other studies. The Xenopus and Drosophila homologues of CGH-1 were proposed to function in maintaining maternally loaded RNAs in a translationally repressed state until they are needed during the early rapid embryonic divisions (Ladomery et al., 1997; Minshall et al., 2001; Nakamura et al., 2001). If CAR-1 represses translation, the cytokinesis defect in car-1inhibited embryos could result from the inappropriate translation of a specific set of maternally contributed RNAs. Alternatively, CAR-1 could play a role in activating the translation of a subset of maternally loaded RNAs. In this scenario, depletion of critical target RNAs might recapitulate the CAR-1 depletion phenotype. Identification of the relevant targets is necessary to understand further the connection between this conserved RNP complex and embryonic cytokinesis.
A novel role for interzonal microtubule bundles during asymmetric cytokinesis
Our analysis of furrow dynamics using a GFP-fusion that specifically localizes to the plasma membrane highlights the asymmetric nature of cytokinesis in wild-type C. elegans embryos (see Fig. 9 for schematic). When viewed in cross section, a primary furrow first comes in from one side of the embryo (Fig. 9 A; top panels). When the primary furrow encounters the interzonal microtubule bundles between the separating chromosomes, a transition occurs in which ingression of the primary furrow slows, and a secondary furrow begins to come in from the opposite side of the embryo (Fig. 9 A; bottom panels). In CAR-1depleted embryos, which lack interzonal microtubule bundles, a primary furrow initiates ingression from one side of the embryo with a rate similar to the wild type. However, the transition to ingression from the opposite side of the embryo never occurs (Fig. 9 B). Consequently, the contractile ring frequently fails to close and the furrow regresses.
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Materials and methods |
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dsRNA and antibody production
Oligonucleotides that were used for double-stranded (dsRNA) production are listed in Table SI A. DNA templates for dsRNA synthesis were amplified from N2 genomic DNA. To generate antibodies directed against CAR-1, the final 300 nucleotides of Y18D10a.17 were amplified from cDNA yk729c6 (obtained from Y. Kohara, National Institute of Genetics, Mishima, Japan) and inserted into pGEX6P-1 (GE Healthcare). The purified GST fusion protein was outsourced for injection into rabbits (Covance), and affinity purification of CAR-1 antibodies was performed as described previously, after removal of the GST by cleavage of the antigen with Prescission protease (Desai et al., 2003). Purified antibodies were labeled directly with a Cy3 fluorescent dye as described (Francis-Lang et al., 1999).
RNAi, blotting of dsRNA-injected worms, and brood size determination
L4 hermaphrodites were injected with dsRNA and were incubated at 20°C for 45 h before analysis. For partial depletions, L4 hermaphrodites were incubated for 2224 h at 20°C after injection of dsRNA. Immunoblotting after RNAi was performed as described (Desai et al., 2003). To determine brood size, injected or control worms were moved every 12 h to individual plates, and the total number of embryos laid on the plate was counted for each period. For each time interval, percent hatching was determined by calculating the number of viable L1 progeny and dividing by the total number of embryos on that plate.
Microscopy and kymograph construction
For analysis of fixed samples mounted in PPDM (90% glycerol, 0.5% p-phenylenediamine, 20 mM Tris-HCl pH 8.8), images were acquired on a DeltaVision deconvolution Olympus IX70 microscope (Applied Precision) equipped with a CoolSnap CCD camera (Roper Scientific) at 20°C using a 100x, 1.35 NA Olympus U-Planapo oil objective lens. Immunofluorescence of fixed embryos was performed as described (Desai et al., 2003), using the following rabbit antibodies at a concentration of 1 µg/ml: CAR-1 (Cy3-labeled; described above);
AIR-2 (Cy-5 labeled; generated against a GST fusion to the full-length protein);
ZEN-4 (Cy-5 labeled; generated against a GST fusion to the COOH-terminal 108 aa); the mouse monoclonal antibody DM1
(Oregon green 488labeled; Sigma-Aldrich); the goat polyclonal GFP antibody (Oregon green 488labeled; generated against a 6x-histidine fusion to the full- length protein); and the unlabeled rat CGH-1 antibody (JDCR5; a gift of K. Blackwell, Joslin Diabetes Center, Boston, MA). For analysis of gonads, the tails of adult hermaphrodites were amputated in 5% sucrose and 100 mM NaCl to extrude the gonads. Fixation and immunofluorescence on gonads was performed as described for embryos. For live analysis, embryos were mounted on agarose pads as described previously (Oegema et al., 2001), and imaged on a spinning disc confocal microscope (Nikon Eclipse TE2000-E) equipped with a Hamamatsu Orca-ER CCD camera at 20°C using a Nikon 60x, 1.4 NA Planapo oil objective lens. For osmotically sensitive embryos and embryos imaged in the absence of compression, filming was performed in a depression slide containing meiosis media (25 mM Hepes at pH 7.4, 60% Leibowitz L-15 Media, 20% FBS, 500 µg/ml inulin) and sealed with petroleum jelly. Analysis of spindle pole separation, spindle microtubule density, and furrow movement was performed using Metamorph software.
Kymographs were constructed by compressing the image of the furrow region from each time point (same region as in Fig. 5 B) to a single vertical line, in which the maximum intensity along the x-axis of each original image is displayed for each point along the y-axis. The vertical strips for sequential time points are laid adjacent to each other so that time increases from left to right along the x-axis.
GFPLAP purification, mass spectrometry, and immunoprecipitation
Adult hermaphrodites expressing GFPLAP:CAR-1 were grown synchronously in liquid culture (Cheeseman et al., 2004), washed in lysis buffer (50 mM Hepes at pH 7.4, 1 mM EDTA, 1 mM MgCl2, 100 mM KCl, and 10% glycerol), and drop frozen in liquid N2. Extracts were generated, and CAR-1 interacting proteins were isolated as described (Cheeseman et al., 2004). Mass spectrometry was performed as described (Cheeseman et al., 2004), using the most recent version of the predicted C. elegans proteins (Wormpep111).
Immunoprecipitations were performed as described previously (Desai et al., 2003), except that extracts were incubated for 20 min in the presence or absence of 5 µg/ml RNaseA at room temperature before immunoprecipitation.
Online supplemental material
Fig. S1 shows that both the CAR-1 Sm domain and RGG box bind to RNA-coated beads. Fig. S2 shows that CAR-1 and CGH-1 both localize to P-granules and smaller cytoplasmic particles. Fig. S3 depicts how microtubule density is reduced in the region between the centrosome and the chromosomes in metaphase spindles from CAR-1depleted embryos. Fig. S4 shows the absence of inter-zonal microtubules following car-1(RNAi) results in a defect in ZEN-4 localization. Video 1: the first mitotic division of a wild-type C. elegans embryo filmed using DIC microscopy. Video 2: the first mitotic division of an embryo laid by a car-1(RNAi) hermaphrodite (RNA #194) filmed by DIC microscopy. Video 3: the first mitotic division of an embryo laid by a homozygous car-1(tm1753) hermaphrodite filmed by DIC microscopy. Video 4: the first seven mitotic divisions of an embryo expressing GFPLAP:CAR-1 filmed by spinning disk confocal microscopy. Video 5: the first mitotic division of a wild-type and a car-1(RNAi) (dsRNA #194) embryo expressing GFP:PHPLC11 filmed by spinning disk confocal microscopy. Video 6: the first mitotic division of a wild-type embryo expressing both GFP:
-tubulin and GFP:PHPLC1
1 filmed by spinning disk confocal microscopy. Video 7: the first mitotic division of a wild-type and a car-1(RNAi) embryo (RNA #194) expressing GFP:
-tubulin filmed by spinning disk confocal microscopy. Video 8: the first mitotic division of a wild-type and a car-1(RNAi) embryo (RNA #194) expressing GFP:
-tubulin and GFP:histone H2B filmed by spinning disk confocal microscopy. Video 9: the first mitotic division of a partial cgh-1(RNAi) embryo (RNA #273) expressing GFPLAP:CAR-1.Table S1: dsRNAs and worm strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200506124/DC1.
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Acknowledgments |
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A. Audhya is a Helen Hay Whitney Postdoctoral Fellow. A.S. Maddox is a fellow of the Giannini Family Foundation. K. Oegema is a Pew Scholar in the Biomedical Sciences. A. Desai is a Damon-Runyon Scholar. This work was supported by funding from the Ludwig Institute for Cancer Research to K. Oegema and A. Desai, and National Institutes of Health grant RR11823 that funds the Yeast Resource Center.
Submitted: 21 June 2005
Accepted: 21 September 2005
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