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Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter.

Yang HY, Mains PE, McNally FJ - J. Cell Biol. (2005)

Bottom Line: Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex.After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex.This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
In animals, female meiotic spindles are attached to the egg cortex in a perpendicular orientation at anaphase to allow the selective disposal of three haploid chromosome sets into polar bodies. We have identified a complex of interacting Caenorhabditis elegans proteins that are involved in the earliest step in asymmetric positioning of anastral meiotic spindles, translocation to the cortex. This complex is composed of the kinesin-1 heavy chain orthologue, UNC-116, the kinesin light chain orthologues, KLC-1 and -2, and a novel cargo adaptor, KCA-1. Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex. After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex. This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.

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C10H11.10/KCA-1 is required for normal translocation of the meiotic spindle to the cortex. (A) Images of GFP-tubulin fluorescence are shown from a representative time-lapse sequence from a meiotic embryo within a kca-1(RNAi) worm. The cell cortex was highlighted in each image for clarity. The meiosis I and meiosis II spindles do not move toward the cortex until after spindle shortening has initiated. The asterisk indicates exit from the spermatheca. (B) Fixed time point image of mat-2(ts); kca-1(RNAi) worms shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
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fig4: C10H11.10/KCA-1 is required for normal translocation of the meiotic spindle to the cortex. (A) Images of GFP-tubulin fluorescence are shown from a representative time-lapse sequence from a meiotic embryo within a kca-1(RNAi) worm. The cell cortex was highlighted in each image for clarity. The meiosis I and meiosis II spindles do not move toward the cortex until after spindle shortening has initiated. The asterisk indicates exit from the spermatheca. (B) Fixed time point image of mat-2(ts); kca-1(RNAi) worms shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.

Mentions: In neurons, a membrane-associated cargo-adaptor protein called sunday driver forms a bridge between kinesin light chains and vesicles transported during fast axonal transport (Bowman et al., 2000). To identify a possible cargo-adaptor protein linking KLC-1 or KLC-2 to the meiotic spindle, we used RNAi to screen putative kinesin light chain interacting proteins identified in a large scale yeast two hybrid study (Li et al., 2004). Treatment of mat-2(ts), GFP-tubulin worms with dsRNA corresponding to one of these interacting gene products (C10H11.10), resulted in meiotic spindles that were far from the cortex of metaphase-arrested embryos (Fig. 4 B). In contrast, all meiotic spindles were at the cortex of metaphase-arrested embryos of mat-2(ts), GFP-tubulin worms treated with other dsRNAs (unpublished data). Time-lapse imaging of GFP-tubulin in 11/11 C10H11.10(RNAi) single mutant worms revealed a block in preanaphase spindle translocation identical to that observed in unc-116(RNAi) or klc-1(RNAi); klc-2(RNAi) worms (Fig. 4 A and Table I). C10H11.10(RNAi) worms also exhibited embryonic lethality (Table II) and polar body defects (Table III) similar to those seen in unc-116(RNAi) and klc-1(RNAi); klc-2(RNAi) worms. These data are consistent with a mechanism in which the C10H11.10 gene product forms a bridge between the UNC-116–KLC-1,2 kinesin and the meiotic spindle. We have therefore given C10H11.10 the name kca-1, for kinesin cargo adaptor.


Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter.

Yang HY, Mains PE, McNally FJ - J. Cell Biol. (2005)

C10H11.10/KCA-1 is required for normal translocation of the meiotic spindle to the cortex. (A) Images of GFP-tubulin fluorescence are shown from a representative time-lapse sequence from a meiotic embryo within a kca-1(RNAi) worm. The cell cortex was highlighted in each image for clarity. The meiosis I and meiosis II spindles do not move toward the cortex until after spindle shortening has initiated. The asterisk indicates exit from the spermatheca. (B) Fixed time point image of mat-2(ts); kca-1(RNAi) worms shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
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Related In: Results  -  Collection

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fig4: C10H11.10/KCA-1 is required for normal translocation of the meiotic spindle to the cortex. (A) Images of GFP-tubulin fluorescence are shown from a representative time-lapse sequence from a meiotic embryo within a kca-1(RNAi) worm. The cell cortex was highlighted in each image for clarity. The meiosis I and meiosis II spindles do not move toward the cortex until after spindle shortening has initiated. The asterisk indicates exit from the spermatheca. (B) Fixed time point image of mat-2(ts); kca-1(RNAi) worms shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
Mentions: In neurons, a membrane-associated cargo-adaptor protein called sunday driver forms a bridge between kinesin light chains and vesicles transported during fast axonal transport (Bowman et al., 2000). To identify a possible cargo-adaptor protein linking KLC-1 or KLC-2 to the meiotic spindle, we used RNAi to screen putative kinesin light chain interacting proteins identified in a large scale yeast two hybrid study (Li et al., 2004). Treatment of mat-2(ts), GFP-tubulin worms with dsRNA corresponding to one of these interacting gene products (C10H11.10), resulted in meiotic spindles that were far from the cortex of metaphase-arrested embryos (Fig. 4 B). In contrast, all meiotic spindles were at the cortex of metaphase-arrested embryos of mat-2(ts), GFP-tubulin worms treated with other dsRNAs (unpublished data). Time-lapse imaging of GFP-tubulin in 11/11 C10H11.10(RNAi) single mutant worms revealed a block in preanaphase spindle translocation identical to that observed in unc-116(RNAi) or klc-1(RNAi); klc-2(RNAi) worms (Fig. 4 A and Table I). C10H11.10(RNAi) worms also exhibited embryonic lethality (Table II) and polar body defects (Table III) similar to those seen in unc-116(RNAi) and klc-1(RNAi); klc-2(RNAi) worms. These data are consistent with a mechanism in which the C10H11.10 gene product forms a bridge between the UNC-116–KLC-1,2 kinesin and the meiotic spindle. We have therefore given C10H11.10 the name kca-1, for kinesin cargo adaptor.

Bottom Line: Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex.After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex.This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
In animals, female meiotic spindles are attached to the egg cortex in a perpendicular orientation at anaphase to allow the selective disposal of three haploid chromosome sets into polar bodies. We have identified a complex of interacting Caenorhabditis elegans proteins that are involved in the earliest step in asymmetric positioning of anastral meiotic spindles, translocation to the cortex. This complex is composed of the kinesin-1 heavy chain orthologue, UNC-116, the kinesin light chain orthologues, KLC-1 and -2, and a novel cargo adaptor, KCA-1. Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex. After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex. This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.

Show MeSH