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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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The atypical Sm domain is not required for CAR-1 localization but is essential for its function. (A) Schematic illustrations of the domain structure of two fusion proteins whose functionality was compared. A GFP-containing localization and purification (GFPLAP) tag was fused to full-length CAR-1 (top) or a truncated version of CAR-1 lacking the NH2-terminal Sm domain (bottom). The S-peptide sequence, a component of the GFPLAP tag used for biochemical purification, also is indicated. (B) Projected three-dimensional dataset of a living wild-type embryo at the four-cell stage expressing GFPLAP:CAR-1. Bar, 10 μm (see also Video 4). (C) Single section spinning disc confocal images of living prometaphase embryos expressing GFPLAP:CAR-1 (left) or GFPLAP:CAR-1ΔN (right) are shown after depletion of endogenous CAR-1 using car-1 3′UTR RNAi. Bar, 10 μm. Western blots of extracts prepared from GFPLAP:CAR-1–expressing worms (D) or GFPLAP:CAR-1ΔN–expressing worms (E) that have been depleted specifically of endogenous CAR-1 by RNAi against the car-1 3′UTR. Serial dilutions of extracts prepared from untreated worms expressing GFPLAP:CAR-1 or GFPLAP:CAR-1ΔN were loaded to quantify depletion levels. (F) Wild-type (N2), GFPLAP:CAR-1–expressing, and GFPLAP:CAR-1ΔN–expressing hermaphrodites that were injected with dsRNA targeted against the car-1 3′UTR to deplete the endogenous protein were scored for brood size and embryonic lethality.
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fig3: The atypical Sm domain is not required for CAR-1 localization but is essential for its function. (A) Schematic illustrations of the domain structure of two fusion proteins whose functionality was compared. A GFP-containing localization and purification (GFPLAP) tag was fused to full-length CAR-1 (top) or a truncated version of CAR-1 lacking the NH2-terminal Sm domain (bottom). The S-peptide sequence, a component of the GFPLAP tag used for biochemical purification, also is indicated. (B) Projected three-dimensional dataset of a living wild-type embryo at the four-cell stage expressing GFPLAP:CAR-1. Bar, 10 μm (see also Video 4). (C) Single section spinning disc confocal images of living prometaphase embryos expressing GFPLAP:CAR-1 (left) or GFPLAP:CAR-1ΔN (right) are shown after depletion of endogenous CAR-1 using car-1 3′UTR RNAi. Bar, 10 μm. Western blots of extracts prepared from GFPLAP:CAR-1–expressing worms (D) or GFPLAP:CAR-1ΔN–expressing worms (E) that have been depleted specifically of endogenous CAR-1 by RNAi against the car-1 3′UTR. Serial dilutions of extracts prepared from untreated worms expressing GFPLAP:CAR-1 or GFPLAP:CAR-1ΔN were loaded to quantify depletion levels. (F) Wild-type (N2), GFPLAP:CAR-1–expressing, and GFPLAP:CAR-1ΔN–expressing hermaphrodites that were injected with dsRNA targeted against the car-1 3′UTR to deplete the endogenous protein were scored for brood size and embryonic lethality.

Mentions: To study its role in cytokinesis, we injected hermaphrodites with double-stranded RNA (dsRNA) against car-1, and analyzed embryos that were laid by the injected mothers, which are depleted of maternally loaded CAR-1 protein. To ensure specificity, dsRNAs against three different regions of the car-1 gene were tested (Table S1 A; available at http://www.jcb.org/cgi/content/full/jcb.200506124/DC1). 45 h after injection, when Western blotting revealed that CAR-1 was >95% depleted (e.g., see Fig. 3 D), >99% embryonic lethality was observed (e.g., see Fig. 3 F). Analysis of the depleted embryos by DIC confirmed the cytokinesis defect that was reported by Zipperlen and coworkers (2001) (Fig. 1 B; see Videos 1 and 2). In a minority of cases (n = 9/50), the first cytokinesis succeeded, but subsequent divisions failed, which accounted for the penetrant embryonic lethality.


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)

The atypical Sm domain is not required for CAR-1 localization but is essential for its function. (A) Schematic illustrations of the domain structure of two fusion proteins whose functionality was compared. A GFP-containing localization and purification (GFPLAP) tag was fused to full-length CAR-1 (top) or a truncated version of CAR-1 lacking the NH2-terminal Sm domain (bottom). The S-peptide sequence, a component of the GFPLAP tag used for biochemical purification, also is indicated. (B) Projected three-dimensional dataset of a living wild-type embryo at the four-cell stage expressing GFPLAP:CAR-1. Bar, 10 μm (see also Video 4). (C) Single section spinning disc confocal images of living prometaphase embryos expressing GFPLAP:CAR-1 (left) or GFPLAP:CAR-1ΔN (right) are shown after depletion of endogenous CAR-1 using car-1 3′UTR RNAi. Bar, 10 μm. Western blots of extracts prepared from GFPLAP:CAR-1–expressing worms (D) or GFPLAP:CAR-1ΔN–expressing worms (E) that have been depleted specifically of endogenous CAR-1 by RNAi against the car-1 3′UTR. Serial dilutions of extracts prepared from untreated worms expressing GFPLAP:CAR-1 or GFPLAP:CAR-1ΔN were loaded to quantify depletion levels. (F) Wild-type (N2), GFPLAP:CAR-1–expressing, and GFPLAP:CAR-1ΔN–expressing hermaphrodites that were injected with dsRNA targeted against the car-1 3′UTR to deplete the endogenous protein were scored for brood size and embryonic lethality.
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fig3: The atypical Sm domain is not required for CAR-1 localization but is essential for its function. (A) Schematic illustrations of the domain structure of two fusion proteins whose functionality was compared. A GFP-containing localization and purification (GFPLAP) tag was fused to full-length CAR-1 (top) or a truncated version of CAR-1 lacking the NH2-terminal Sm domain (bottom). The S-peptide sequence, a component of the GFPLAP tag used for biochemical purification, also is indicated. (B) Projected three-dimensional dataset of a living wild-type embryo at the four-cell stage expressing GFPLAP:CAR-1. Bar, 10 μm (see also Video 4). (C) Single section spinning disc confocal images of living prometaphase embryos expressing GFPLAP:CAR-1 (left) or GFPLAP:CAR-1ΔN (right) are shown after depletion of endogenous CAR-1 using car-1 3′UTR RNAi. Bar, 10 μm. Western blots of extracts prepared from GFPLAP:CAR-1–expressing worms (D) or GFPLAP:CAR-1ΔN–expressing worms (E) that have been depleted specifically of endogenous CAR-1 by RNAi against the car-1 3′UTR. Serial dilutions of extracts prepared from untreated worms expressing GFPLAP:CAR-1 or GFPLAP:CAR-1ΔN were loaded to quantify depletion levels. (F) Wild-type (N2), GFPLAP:CAR-1–expressing, and GFPLAP:CAR-1ΔN–expressing hermaphrodites that were injected with dsRNA targeted against the car-1 3′UTR to deplete the endogenous protein were scored for brood size and embryonic lethality.
Mentions: To study its role in cytokinesis, we injected hermaphrodites with double-stranded RNA (dsRNA) against car-1, and analyzed embryos that were laid by the injected mothers, which are depleted of maternally loaded CAR-1 protein. To ensure specificity, dsRNAs against three different regions of the car-1 gene were tested (Table S1 A; available at http://www.jcb.org/cgi/content/full/jcb.200506124/DC1). 45 h after injection, when Western blotting revealed that CAR-1 was >95% depleted (e.g., see Fig. 3 D), >99% embryonic lethality was observed (e.g., see Fig. 3 F). Analysis of the depleted embryos by DIC confirmed the cytokinesis defect that was reported by Zipperlen and coworkers (2001) (Fig. 1 B; see Videos 1 and 2). In a minority of cases (n = 9/50), the first cytokinesis succeeded, but subsequent divisions failed, which accounted for the penetrant embryonic lethality.

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