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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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Kinesin light chains are required for normal translocation of the meiotic spindle to the cortex. Images of GFP-tubulin fluorescence are shown from representative time-lapse sequences of a meiotic embryo within a klc-1(RNAi) worm (A) and a klc-1(RNAi); klc-2(RNAi) worm (B). The cell cortex was highlighted in each image for clarity. In both cases, the meiosis I and II spindles do not move toward the cortex until after spindle shortening has initiated. Asterisks indicate exit from the spermatheca. (C) Fixed time point image of a mat-2(ts); klc-1(RNAi); klc-2(RNAi) triple mutant worm shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
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fig3: Kinesin light chains are required for normal translocation of the meiotic spindle to the cortex. Images of GFP-tubulin fluorescence are shown from representative time-lapse sequences of a meiotic embryo within a klc-1(RNAi) worm (A) and a klc-1(RNAi); klc-2(RNAi) worm (B). The cell cortex was highlighted in each image for clarity. In both cases, the meiosis I and II spindles do not move toward the cortex until after spindle shortening has initiated. Asterisks indicate exit from the spermatheca. (C) Fixed time point image of a mat-2(ts); klc-1(RNAi); klc-2(RNAi) triple mutant worm shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.

Mentions: Kinesin-1 purified from a variety of species, including C. elegans, consists of a tetramer with two heavy chain and two light chain subunits (Vale et al., 1985; Saxton et al., 1988; Signor et al., 1999). To test whether or not early meiotic spindle translocation depends on a kinesin-1 heavy chain/light chain complex, we acquired GFP-tubulin time-lapse sequences from worms treated with dsRNA corresponding to either of the C. elegans kinesin light chain homologues, KLC-1 or KLC-2. A block to early spindle translocation was observed in 3/6 klc-1(RNAi) worms, 2/7 klc-2(RNAi) worms, and 4/5 doubly treated klc-1(RNAi); klc-2(RNAi) worms (Fig. 3, A and B; and Table I). In addition, depletion of kinesin light chains in metaphase-arrested mat-2(ts) embryos resulted in spindles arrested far from the cortex (Fig. 3 C). Thus, the phenotype of klc-1(RNAi) or klc-2(RNAi) worms was qualitatively the same, but quantitatively weaker, than the phenotype of unc-116(RNAi) embryos. Western blots of klc-2(RNAi) worms probed with a KLC-2–specific antibody revealed that considerable KLC-2 protein product remains in these worms (Fig. S1). Thus the weaker phenotypes observed were at least in part due to the incomplete effectiveness of the RNAi. Simultaneous treatment of worms with dsRNAs corresponding to both light chains (klc-1(RNAi); klc-2(RNAi)) resulted in embryonic lethality (Table II), polar body defects (Table III), and translocation defects that were quantitatively similar to unc-116(RNAi) worms. These results indicate that the two kinesin light chains act redundantly and that a complex of kinesin heavy and light chains is essential for the preanaphase translocation of the meiotic spindle.


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)

Kinesin light chains are required for normal translocation of the meiotic spindle to the cortex. Images of GFP-tubulin fluorescence are shown from representative time-lapse sequences of a meiotic embryo within a klc-1(RNAi) worm (A) and a klc-1(RNAi); klc-2(RNAi) worm (B). The cell cortex was highlighted in each image for clarity. In both cases, the meiosis I and II spindles do not move toward the cortex until after spindle shortening has initiated. Asterisks indicate exit from the spermatheca. (C) Fixed time point image of a mat-2(ts); klc-1(RNAi); klc-2(RNAi) triple mutant worm shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
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Related In: Results  -  Collection

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fig3: Kinesin light chains are required for normal translocation of the meiotic spindle to the cortex. Images of GFP-tubulin fluorescence are shown from representative time-lapse sequences of a meiotic embryo within a klc-1(RNAi) worm (A) and a klc-1(RNAi); klc-2(RNAi) worm (B). The cell cortex was highlighted in each image for clarity. In both cases, the meiosis I and II spindles do not move toward the cortex until after spindle shortening has initiated. Asterisks indicate exit from the spermatheca. (C) Fixed time point image of a mat-2(ts); klc-1(RNAi); klc-2(RNAi) triple mutant worm shows a meiotic spindle arrested far from the cortex. Bars, 10 μm.
Mentions: Kinesin-1 purified from a variety of species, including C. elegans, consists of a tetramer with two heavy chain and two light chain subunits (Vale et al., 1985; Saxton et al., 1988; Signor et al., 1999). To test whether or not early meiotic spindle translocation depends on a kinesin-1 heavy chain/light chain complex, we acquired GFP-tubulin time-lapse sequences from worms treated with dsRNA corresponding to either of the C. elegans kinesin light chain homologues, KLC-1 or KLC-2. A block to early spindle translocation was observed in 3/6 klc-1(RNAi) worms, 2/7 klc-2(RNAi) worms, and 4/5 doubly treated klc-1(RNAi); klc-2(RNAi) worms (Fig. 3, A and B; and Table I). In addition, depletion of kinesin light chains in metaphase-arrested mat-2(ts) embryos resulted in spindles arrested far from the cortex (Fig. 3 C). Thus, the phenotype of klc-1(RNAi) or klc-2(RNAi) worms was qualitatively the same, but quantitatively weaker, than the phenotype of unc-116(RNAi) embryos. Western blots of klc-2(RNAi) worms probed with a KLC-2–specific antibody revealed that considerable KLC-2 protein product remains in these worms (Fig. S1). Thus the weaker phenotypes observed were at least in part due to the incomplete effectiveness of the RNAi. Simultaneous treatment of worms with dsRNAs corresponding to both light chains (klc-1(RNAi); klc-2(RNAi)) resulted in embryonic lethality (Table II), polar body defects (Table III), and translocation defects that were quantitatively similar to unc-116(RNAi) worms. These results indicate that the two kinesin light chains act redundantly and that a complex of kinesin heavy and light chains is essential for the preanaphase translocation of the meiotic spindle.

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