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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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Disruption of cleavage furrow ingression in CAR-1–depleted embryos. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:PHPLC1δ1, which localizes to the plasma membrane. Time in seconds after initiation of furrowing is indicated in the lower right corner of each panel. (See also Video 5.) Bar, 10 μm. Arrows highlight the failure of polar body extrusion. Arrowheads point to secondary furrow, which fails to ingress in CAR-1–depleted embryos. (B) Same as in A, except only a 7.3-μm wide vertical section containing the furrow is shown for each time point. Bar, 5 μm. (C) Kymographs of the regions shown in B, comparing furrow ingression in wild-type and CAR-1–depleted embryos (see Methods and materials for details on kymograph construction). The slope of the yellow line indicates the initial rate of primary furrow ingression, which is identical in wild-type and CAR-1–depleted embryos. In wild-type embryos, the primary furrow encounters the midbody and ceases to ingress (time indicated by red dashed line). At a similar time in CAR-1–depleted embryos, the primary furrow slows (cyan line) but does not stop. In wild-type embryos, a secondary furrow begins to ingress from the opposite side of the embryo (pink line) as the primary furrow ceases its inward movement. In CAR-1–depleted embryos, no ingression of a secondary furrow is observed. Bar, 5 μm.
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fig5: Disruption of cleavage furrow ingression in CAR-1–depleted embryos. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:PHPLC1δ1, which localizes to the plasma membrane. Time in seconds after initiation of furrowing is indicated in the lower right corner of each panel. (See also Video 5.) Bar, 10 μm. Arrows highlight the failure of polar body extrusion. Arrowheads point to secondary furrow, which fails to ingress in CAR-1–depleted embryos. (B) Same as in A, except only a 7.3-μm wide vertical section containing the furrow is shown for each time point. Bar, 5 μm. (C) Kymographs of the regions shown in B, comparing furrow ingression in wild-type and CAR-1–depleted embryos (see Methods and materials for details on kymograph construction). The slope of the yellow line indicates the initial rate of primary furrow ingression, which is identical in wild-type and CAR-1–depleted embryos. In wild-type embryos, the primary furrow encounters the midbody and ceases to ingress (time indicated by red dashed line). At a similar time in CAR-1–depleted embryos, the primary furrow slows (cyan line) but does not stop. In wild-type embryos, a secondary furrow begins to ingress from the opposite side of the embryo (pink line) as the primary furrow ceases its inward movement. In CAR-1–depleted embryos, no ingression of a secondary furrow is observed. Bar, 5 μm.

Mentions: To analyze the cytokinesis defect in CAR-1–depleted embryos, we constructed a C. elegans strain expressing a fusion of GFP with a pleckstrin homology (PH) domain derived from mammalian PLC1δ1 to monitor cleavage furrow ingression (Fig. 5). The PLC1δ1 PH domain binds with high affinity to a phosphoinositide lipid (PI4,5P2) that is generated on the plasma membrane in most cell types (for review see Hurley and Meyer, 2001). Imaging of GFP:PHPLC1δ1 in wild-type embryos revealed a striking asymmetry in cleavage furrow ingression. A primary furrow was observed initially coming in from one side of the embryo (yellow line on kymograph in Fig. 5 C; Video 5). As the primary furrow ceased its ingression, a secondary furrow began to come in from the opposite side of the embryo (pink line on kymograph in Fig. 5 C). Simultaneous visualization of microtubules and the plasma membrane (Video 6) revealed that the secondary furrow began to ingress when the primary furrow came into contact with the interzonal microtubule bundles that form between the separating chromosomes (see Fig. 9 for a schematic of this transition). After ingression of the secondary furrow slows, a small gap remains that gradually closes (Fig. 5, B and C). This asymmetric ingression is not a consequence of embryo compression (unpublished data).


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)

Disruption of cleavage furrow ingression in CAR-1–depleted embryos. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:PHPLC1δ1, which localizes to the plasma membrane. Time in seconds after initiation of furrowing is indicated in the lower right corner of each panel. (See also Video 5.) Bar, 10 μm. Arrows highlight the failure of polar body extrusion. Arrowheads point to secondary furrow, which fails to ingress in CAR-1–depleted embryos. (B) Same as in A, except only a 7.3-μm wide vertical section containing the furrow is shown for each time point. Bar, 5 μm. (C) Kymographs of the regions shown in B, comparing furrow ingression in wild-type and CAR-1–depleted embryos (see Methods and materials for details on kymograph construction). The slope of the yellow line indicates the initial rate of primary furrow ingression, which is identical in wild-type and CAR-1–depleted embryos. In wild-type embryos, the primary furrow encounters the midbody and ceases to ingress (time indicated by red dashed line). At a similar time in CAR-1–depleted embryos, the primary furrow slows (cyan line) but does not stop. In wild-type embryos, a secondary furrow begins to ingress from the opposite side of the embryo (pink line) as the primary furrow ceases its inward movement. In CAR-1–depleted embryos, no ingression of a secondary furrow is observed. Bar, 5 μm.
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Related In: Results  -  Collection

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

fig5: Disruption of cleavage furrow ingression in CAR-1–depleted embryos. (A) Selected panels from time-lapse sequences of wild-type (left column) and CAR-1–depleted (right column) embryos expressing GFP:PHPLC1δ1, which localizes to the plasma membrane. Time in seconds after initiation of furrowing is indicated in the lower right corner of each panel. (See also Video 5.) Bar, 10 μm. Arrows highlight the failure of polar body extrusion. Arrowheads point to secondary furrow, which fails to ingress in CAR-1–depleted embryos. (B) Same as in A, except only a 7.3-μm wide vertical section containing the furrow is shown for each time point. Bar, 5 μm. (C) Kymographs of the regions shown in B, comparing furrow ingression in wild-type and CAR-1–depleted embryos (see Methods and materials for details on kymograph construction). The slope of the yellow line indicates the initial rate of primary furrow ingression, which is identical in wild-type and CAR-1–depleted embryos. In wild-type embryos, the primary furrow encounters the midbody and ceases to ingress (time indicated by red dashed line). At a similar time in CAR-1–depleted embryos, the primary furrow slows (cyan line) but does not stop. In wild-type embryos, a secondary furrow begins to ingress from the opposite side of the embryo (pink line) as the primary furrow ceases its inward movement. In CAR-1–depleted embryos, no ingression of a secondary furrow is observed. Bar, 5 μm.
Mentions: To analyze the cytokinesis defect in CAR-1–depleted embryos, we constructed a C. elegans strain expressing a fusion of GFP with a pleckstrin homology (PH) domain derived from mammalian PLC1δ1 to monitor cleavage furrow ingression (Fig. 5). The PLC1δ1 PH domain binds with high affinity to a phosphoinositide lipid (PI4,5P2) that is generated on the plasma membrane in most cell types (for review see Hurley and Meyer, 2001). Imaging of GFP:PHPLC1δ1 in wild-type embryos revealed a striking asymmetry in cleavage furrow ingression. A primary furrow was observed initially coming in from one side of the embryo (yellow line on kymograph in Fig. 5 C; Video 5). As the primary furrow ceased its ingression, a secondary furrow began to come in from the opposite side of the embryo (pink line on kymograph in Fig. 5 C). Simultaneous visualization of microtubules and the plasma membrane (Video 6) revealed that the secondary furrow began to ingress when the primary furrow came into contact with the interzonal microtubule bundles that form between the separating chromosomes (see Fig. 9 for a schematic of this transition). After ingression of the secondary furrow slows, a small gap remains that gradually closes (Fig. 5, B and C). This asymmetric ingression is not a consequence of embryo compression (unpublished data).

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