Limits...
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.

Show MeSH

Related in: MedlinePlus

CAR-1 depletion results in a pronounced defect in anaphase spindle structure. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:α-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows indicate the region between the segregated chromosomes where interzonal microtubule bundles normally form (see also Video 7). Bar, 10 μm. (B) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows point to chromosome bridges that become evident after anaphase onset in CAR-1–depleted embryos. (See also Video 8.) Bar, 10 μm. (C) The distance between spindle poles was tracked for 15 wild-type (WT) and 19 CAR-1–depleted embryos imaged as in A. Average pole-to-pole distance is plotted versus time after NEBD (nuclear envelope breakdown). Error bars represent the SEM with a confidence interval of 0.95. For reference, the time of onset of chromosome segregation is indicated. Embryos in which extrusion of the second polar body failed and extra chromatin was present in the mitotic spindle (n = 6/50) were not used for the analysis of spindle length.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171198&req=5

fig6: CAR-1 depletion results in a pronounced defect in anaphase spindle structure. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:α-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows indicate the region between the segregated chromosomes where interzonal microtubule bundles normally form (see also Video 7). Bar, 10 μm. (B) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows point to chromosome bridges that become evident after anaphase onset in CAR-1–depleted embryos. (See also Video 8.) Bar, 10 μm. (C) The distance between spindle poles was tracked for 15 wild-type (WT) and 19 CAR-1–depleted embryos imaged as in A. Average pole-to-pole distance is plotted versus time after NEBD (nuclear envelope breakdown). Error bars represent the SEM with a confidence interval of 0.95. For reference, the time of onset of chromosome segregation is indicated. Embryos in which extrusion of the second polar body failed and extra chromatin was present in the mitotic spindle (n = 6/50) were not used for the analysis of spindle length.

Mentions: To analyze spindle structure directly, we examined embryos expressing GFP:α-tubulin or coexpressing GFP-histone H2B and GFP:γ-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-1–depleted embryos (n = 45/50) (Fig. 6 B, 20/40-s panels; Video 8). At anaphase onset, a dramatic defect was apparent in CAR-1–depleted 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-1–depleted embryos.


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 depletion results in a pronounced defect in anaphase spindle structure. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:α-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows indicate the region between the segregated chromosomes where interzonal microtubule bundles normally form (see also Video 7). Bar, 10 μm. (B) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows point to chromosome bridges that become evident after anaphase onset in CAR-1–depleted embryos. (See also Video 8.) Bar, 10 μm. (C) The distance between spindle poles was tracked for 15 wild-type (WT) and 19 CAR-1–depleted embryos imaged as in A. Average pole-to-pole distance is plotted versus time after NEBD (nuclear envelope breakdown). Error bars represent the SEM with a confidence interval of 0.95. For reference, the time of onset of chromosome segregation is indicated. Embryos in which extrusion of the second polar body failed and extra chromatin was present in the mitotic spindle (n = 6/50) were not used for the analysis of spindle length.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: CAR-1 depletion results in a pronounced defect in anaphase spindle structure. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:α-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows indicate the region between the segregated chromosomes where interzonal microtubule bundles normally form (see also Video 7). Bar, 10 μm. (B) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:histone H2B and GFP:γ-tubulin. Time in seconds after chromosome alignment is indicated in the lower right corner of each panel. Arrows point to chromosome bridges that become evident after anaphase onset in CAR-1–depleted embryos. (See also Video 8.) Bar, 10 μm. (C) The distance between spindle poles was tracked for 15 wild-type (WT) and 19 CAR-1–depleted embryos imaged as in A. Average pole-to-pole distance is plotted versus time after NEBD (nuclear envelope breakdown). Error bars represent the SEM with a confidence interval of 0.95. For reference, the time of onset of chromosome segregation is indicated. Embryos in which extrusion of the second polar body failed and extra chromatin was present in the mitotic spindle (n = 6/50) were not used for the analysis of spindle length.
Mentions: To analyze spindle structure directly, we examined embryos expressing GFP:α-tubulin or coexpressing GFP-histone H2B and GFP:γ-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-1–depleted embryos (n = 45/50) (Fig. 6 B, 20/40-s panels; Video 8). At anaphase onset, a dramatic defect was apparent in CAR-1–depleted 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-1–depleted embryos.

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