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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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AIR-2 and ZEN-4 fail to target to interzonal microtubule bundles in CAR-1–depleted embryos. Simultaneous depletion of GPR-1/2, which inhibits pulling forces on the spindle poles and prevents spindle snapping, does not rescue this defect. (A) Selected images from time-lapse sequences of wild-type, car-1(RNAi), and car-1/gpr-1,2 double RNAi embryos expressing GFP:AIR-2. Only the region of the spindle is shown. Time after chromosome alignment (in seconds) is indicated in the lower right corner of each panel. Bar, 5 μm. Projected three-dimensional datasets of fixed wild-type (B) and CAR-1–depleted (C) embryos stained for DNA, microtubules, AIR-2, and ZEN-4. High magnification panels in the right column are magnified 2.5× relative to the adjacent images. The ZEN-4 high magnification image is 10× overexposed. Bar, 10 μm.
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fig7: AIR-2 and ZEN-4 fail to target to interzonal microtubule bundles in CAR-1–depleted embryos. Simultaneous depletion of GPR-1/2, which inhibits pulling forces on the spindle poles and prevents spindle snapping, does not rescue this defect. (A) Selected images from time-lapse sequences of wild-type, car-1(RNAi), and car-1/gpr-1,2 double RNAi embryos expressing GFP:AIR-2. Only the region of the spindle is shown. Time after chromosome alignment (in seconds) is indicated in the lower right corner of each panel. Bar, 5 μm. Projected three-dimensional datasets of fixed wild-type (B) and CAR-1–depleted (C) embryos stained for DNA, microtubules, AIR-2, and ZEN-4. High magnification panels in the right column are magnified 2.5× relative to the adjacent images. The ZEN-4 high magnification image is 10× overexposed. Bar, 10 μm.

Mentions: In CAR-1–depleted embryos, interzonal microtubule bundles are absent or reduced significantly. This defect is specific, because it is not seen after a variety of perturbations that destabilize the microtubule cytoskeleton, such as nocodazole treatment or depletion of the microtubule-stabilizing protein, ZYG-9 (Albertson, 1984; Matthews et al., 1998). The formation of interzonal microtubule bundles requires two protein complexes: the chromosomal passenger complex, which includes the Aurora B kinase, AIR-2 (reviewed in Vagnarelli and Earnshaw, 2004), and centralspindlin, a complex that contains the plus-end–directed kinesin, ZEN-4 (for review see Glotzer, 2003). Both complexes are recruited to interzonal microtubule bundles as they form, and therefore, are excellent markers for these structures. To examine the dynamics of both complexes, we used strains expressing GFP fusions with AIR-2 and ZEN-4. In wild-type embryos, GFP:AIR-2 localizes to chromosomes at metaphase (Fig. 7 A, 0-s panel). Following chromosome separation, GFP:AIR-2 is found on chromosomes and interzonal microtubule bundles. In CAR-1–depleted embryos, GFP:AIR-2 was present on mitotic chromosomes as in the wild type. However, after chromosome separation, GFP:AIR-2 remained exclusively localized to chromosomes (Fig. 7A). Similarly, GFP:ZEN-4, which normally accumulates on interzonal microtubule bundles, was essentially absent in CAR-1–depleted embryos (Fig. S4). Identical results were obtained from fixed samples using antibodies to the endogenous proteins (Fig. 7, B and C). The presence of normal levels of Aurora B on chromosomes, and of ZEN-4 on meiotic midbody microtubules in CAR-1–depleted embryos (Fig. 7 C), suggests that that the failure of interzonal microtubule bundle formation in CAR-1–depleted embryos is not due to destabilization of Aurora B or ZEN-4.


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)

AIR-2 and ZEN-4 fail to target to interzonal microtubule bundles in CAR-1–depleted embryos. Simultaneous depletion of GPR-1/2, which inhibits pulling forces on the spindle poles and prevents spindle snapping, does not rescue this defect. (A) Selected images from time-lapse sequences of wild-type, car-1(RNAi), and car-1/gpr-1,2 double RNAi embryos expressing GFP:AIR-2. Only the region of the spindle is shown. Time after chromosome alignment (in seconds) is indicated in the lower right corner of each panel. Bar, 5 μm. Projected three-dimensional datasets of fixed wild-type (B) and CAR-1–depleted (C) embryos stained for DNA, microtubules, AIR-2, and ZEN-4. High magnification panels in the right column are magnified 2.5× relative to the adjacent images. The ZEN-4 high magnification image is 10× overexposed. Bar, 10 μm.
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fig7: AIR-2 and ZEN-4 fail to target to interzonal microtubule bundles in CAR-1–depleted embryos. Simultaneous depletion of GPR-1/2, which inhibits pulling forces on the spindle poles and prevents spindle snapping, does not rescue this defect. (A) Selected images from time-lapse sequences of wild-type, car-1(RNAi), and car-1/gpr-1,2 double RNAi embryos expressing GFP:AIR-2. Only the region of the spindle is shown. Time after chromosome alignment (in seconds) is indicated in the lower right corner of each panel. Bar, 5 μm. Projected three-dimensional datasets of fixed wild-type (B) and CAR-1–depleted (C) embryos stained for DNA, microtubules, AIR-2, and ZEN-4. High magnification panels in the right column are magnified 2.5× relative to the adjacent images. The ZEN-4 high magnification image is 10× overexposed. Bar, 10 μm.
Mentions: In CAR-1–depleted embryos, interzonal microtubule bundles are absent or reduced significantly. This defect is specific, because it is not seen after a variety of perturbations that destabilize the microtubule cytoskeleton, such as nocodazole treatment or depletion of the microtubule-stabilizing protein, ZYG-9 (Albertson, 1984; Matthews et al., 1998). The formation of interzonal microtubule bundles requires two protein complexes: the chromosomal passenger complex, which includes the Aurora B kinase, AIR-2 (reviewed in Vagnarelli and Earnshaw, 2004), and centralspindlin, a complex that contains the plus-end–directed kinesin, ZEN-4 (for review see Glotzer, 2003). Both complexes are recruited to interzonal microtubule bundles as they form, and therefore, are excellent markers for these structures. To examine the dynamics of both complexes, we used strains expressing GFP fusions with AIR-2 and ZEN-4. In wild-type embryos, GFP:AIR-2 localizes to chromosomes at metaphase (Fig. 7 A, 0-s panel). Following chromosome separation, GFP:AIR-2 is found on chromosomes and interzonal microtubule bundles. In CAR-1–depleted embryos, GFP:AIR-2 was present on mitotic chromosomes as in the wild type. However, after chromosome separation, GFP:AIR-2 remained exclusively localized to chromosomes (Fig. 7A). Similarly, GFP:ZEN-4, which normally accumulates on interzonal microtubule bundles, was essentially absent in CAR-1–depleted embryos (Fig. S4). Identical results were obtained from fixed samples using antibodies to the endogenous proteins (Fig. 7, B and C). The presence of normal levels of Aurora B on chromosomes, and of ZEN-4 on meiotic midbody microtubules in CAR-1–depleted embryos (Fig. 7 C), suggests that that the failure of interzonal microtubule bundle formation in CAR-1–depleted embryos is not due to destabilization of Aurora B or ZEN-4.

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