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A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.

Audhya A, Hyndman F, McLeod IX, Maddox AS, Yates JR, Desai A, Oegema K - J. Cell Biol. (2005)

Bottom Line: Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis.This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes.Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA. aaudhya@ucsd.edu

ABSTRACT
Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

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CAR-1 localizes to P-granules and additional smaller cytoplasmic particles. (A) Projected three-dimensional datasets of fixed embryos at the indicated cell cycle stages stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with CAR-1 in green, PGL-1 in red, and DNA in blue also are shown. Panels in the right column are of the boxed area magnified 3× relative to the adjacent images. Arrowheads point to examples of juxtaposed CAR-1– and PGL-1–containing particles during meiosis II. Arrows identify smaller cytoplasmic CAR-1–containing particles that lack PGL-1. Bar, 10 μm. (B) No CAR-1 staining is detected in car-1(RNAi) embryos. Bar, 10 μm.
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fig2: CAR-1 localizes to P-granules and additional smaller cytoplasmic particles. (A) Projected three-dimensional datasets of fixed embryos at the indicated cell cycle stages stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with CAR-1 in green, PGL-1 in red, and DNA in blue also are shown. Panels in the right column are of the boxed area magnified 3× relative to the adjacent images. Arrowheads point to examples of juxtaposed CAR-1– and PGL-1–containing particles during meiosis II. Arrows identify smaller cytoplasmic CAR-1–containing particles that lack PGL-1. Bar, 10 μm. (B) No CAR-1 staining is detected in car-1(RNAi) embryos. Bar, 10 μm.

Mentions: 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.


A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.

Audhya A, Hyndman F, McLeod IX, Maddox AS, Yates JR, Desai A, Oegema K - J. Cell Biol. (2005)

CAR-1 localizes to P-granules and additional smaller cytoplasmic particles. (A) Projected three-dimensional datasets of fixed embryos at the indicated cell cycle stages stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with CAR-1 in green, PGL-1 in red, and DNA in blue also are shown. Panels in the right column are of the boxed area magnified 3× relative to the adjacent images. Arrowheads point to examples of juxtaposed CAR-1– and PGL-1–containing particles during meiosis II. Arrows identify smaller cytoplasmic CAR-1–containing particles that lack PGL-1. Bar, 10 μm. (B) No CAR-1 staining is detected in car-1(RNAi) embryos. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171198&req=5

fig2: CAR-1 localizes to P-granules and additional smaller cytoplasmic particles. (A) Projected three-dimensional datasets of fixed embryos at the indicated cell cycle stages stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with CAR-1 in green, PGL-1 in red, and DNA in blue also are shown. Panels in the right column are of the boxed area magnified 3× relative to the adjacent images. Arrowheads point to examples of juxtaposed CAR-1– and PGL-1–containing particles during meiosis II. Arrows identify smaller cytoplasmic CAR-1–containing particles that lack PGL-1. Bar, 10 μm. (B) No CAR-1 staining is detected in car-1(RNAi) embryos. Bar, 10 μm.
Mentions: 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.

Bottom Line: Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis.This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes.Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA. aaudhya@ucsd.edu

ABSTRACT
Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

Show MeSH
Related in: MedlinePlus