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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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Partial depletion of CGH-1 phenocopies depletion of CAR-1. Selected panels from time-lapse sequence of wild-type (left column) and CGH-1 partially depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin (A), GFP:α-tubulin (B), and GFP:AIR-2 (C). Arrows in B highlight the presence or absence of interzonal microtubules. Arrows in C highlight the presence or absence of AIR-2 on interzonal microtubules; the arrowhead points to polar body chromatin that failed to be extruded properly. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Bar, 10 μm.
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fig8: Partial depletion of CGH-1 phenocopies depletion of CAR-1. Selected panels from time-lapse sequence of wild-type (left column) and CGH-1 partially depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin (A), GFP:α-tubulin (B), and GFP:AIR-2 (C). Arrows in B highlight the presence or absence of interzonal microtubules. Arrows in C highlight the presence or absence of AIR-2 on interzonal microtubules; the arrowhead points to polar body chromatin that failed to be extruded properly. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Bar, 10 μm.

Mentions: 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, 22–24 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-1–depleted embryos were almost identical to those in CAR-1–depleted embryos (Fig. 8). Cytokinesis failed in most partial CGH-1–depleted 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).


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)

Partial depletion of CGH-1 phenocopies depletion of CAR-1. Selected panels from time-lapse sequence of wild-type (left column) and CGH-1 partially depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin (A), GFP:α-tubulin (B), and GFP:AIR-2 (C). Arrows in B highlight the presence or absence of interzonal microtubules. Arrows in C highlight the presence or absence of AIR-2 on interzonal microtubules; the arrowhead points to polar body chromatin that failed to be extruded properly. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171198&req=5

fig8: Partial depletion of CGH-1 phenocopies depletion of CAR-1. Selected panels from time-lapse sequence of wild-type (left column) and CGH-1 partially depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin (A), GFP:α-tubulin (B), and GFP:AIR-2 (C). Arrows in B highlight the presence or absence of interzonal microtubules. Arrows in C highlight the presence or absence of AIR-2 on interzonal microtubules; the arrowhead points to polar body chromatin that failed to be extruded properly. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Bar, 10 μm.
Mentions: 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, 22–24 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-1–depleted embryos were almost identical to those in CAR-1–depleted embryos (Fig. 8). Cytokinesis failed in most partial CGH-1–depleted 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).

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