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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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unc-116(RNAi) spindles are stationary during the period of meiosis when wild-type spindles translocate to the cortex. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from a wild-type worm (A) and an unc-116(RNAi) worm (B). The cell cortex was highlighted in each image and drawings corresponding to each image are included for clarity. (A) The wild-type meiosis I spindle translocated to the cortex from 0 to 0.7 min and adopted an orientation parallel to the cortex until spindle rotation from 8.7 to 9.7 min. (B) In contrast, the meiosis I spindle in the unc-116(RNAi) embryo remained stationary at a position several micrometers from the cortex from 0 to 6.0 min. Spindle shortening initiated just before the start of movement to the cortex (6.0 to 7.2 min) in the unc-116(RNAi) embryo, in contrast with the wild-type spindle, which initiated shortening long after cortical contact. Time 0 indicates exit of the zygote from the spermatheca into the uterus. Corresponding Videos 1 and 2 can be found in the online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1. (C–F) The shortest distance from the edge of the spindle to the cortex (▴) and the pole–pole spindle length (○) was measured in each frame of one representative GFP-tubulin time-lapse sequence from embryos of the indicated genotype. Time 0 is germinal vesicle breakdown. The vertical dotted line indicates the time the embryo exited the spermatheca into the uterus. (C) In wild-type embryos, spindle movement to the cortex initiated before exit from the spermatheca, and the 7.5-μm-long metaphase spindle remained at the cortex for 10 min before initiating its shortening phase. (D) In unc-116(RNAi) embryos, the meiotic spindle did not initiate movement toward the cortex until the time that spindle shortening initiated. (E) In embryos arrested at metaphase I due to a temperature-sensitive APC mutant, mat-2(ts), spindle shortening did not initiate but translocation to the cortex was normal. Thus, wild-type translocation is APC independent. (F) In mat-2(ts); unc-116(RNAi) double mutant embryos, the spindle did not shorten and never translocated to the cortex. Thus, the movement of unc-116(RNAi) spindles toward the cortex is APC dependent. (G and H) Fixed time point images of GFP-tubulin–labeled spindles in metaphase-arrested mat-2(ts) (G) or mat-2(ts); unc-116(RNAi) (H) worms. Bars, 10 μm.
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fig1: unc-116(RNAi) spindles are stationary during the period of meiosis when wild-type spindles translocate to the cortex. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from a wild-type worm (A) and an unc-116(RNAi) worm (B). The cell cortex was highlighted in each image and drawings corresponding to each image are included for clarity. (A) The wild-type meiosis I spindle translocated to the cortex from 0 to 0.7 min and adopted an orientation parallel to the cortex until spindle rotation from 8.7 to 9.7 min. (B) In contrast, the meiosis I spindle in the unc-116(RNAi) embryo remained stationary at a position several micrometers from the cortex from 0 to 6.0 min. Spindle shortening initiated just before the start of movement to the cortex (6.0 to 7.2 min) in the unc-116(RNAi) embryo, in contrast with the wild-type spindle, which initiated shortening long after cortical contact. Time 0 indicates exit of the zygote from the spermatheca into the uterus. Corresponding Videos 1 and 2 can be found in the online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1. (C–F) The shortest distance from the edge of the spindle to the cortex (▴) and the pole–pole spindle length (○) was measured in each frame of one representative GFP-tubulin time-lapse sequence from embryos of the indicated genotype. Time 0 is germinal vesicle breakdown. The vertical dotted line indicates the time the embryo exited the spermatheca into the uterus. (C) In wild-type embryos, spindle movement to the cortex initiated before exit from the spermatheca, and the 7.5-μm-long metaphase spindle remained at the cortex for 10 min before initiating its shortening phase. (D) In unc-116(RNAi) embryos, the meiotic spindle did not initiate movement toward the cortex until the time that spindle shortening initiated. (E) In embryos arrested at metaphase I due to a temperature-sensitive APC mutant, mat-2(ts), spindle shortening did not initiate but translocation to the cortex was normal. Thus, wild-type translocation is APC independent. (F) In mat-2(ts); unc-116(RNAi) double mutant embryos, the spindle did not shorten and never translocated to the cortex. Thus, the movement of unc-116(RNAi) spindles toward the cortex is APC dependent. (G and H) Fixed time point images of GFP-tubulin–labeled spindles in metaphase-arrested mat-2(ts) (G) or mat-2(ts); unc-116(RNAi) (H) worms. Bars, 10 μm.

Mentions: To determine whether or not microtubule motor proteins are involved in the translocation of the meiotic spindle to the oocyte cortex in C. elegans, we analyzed meiotic spindle translocation in worms depleted of different motor subunits by RNAi. There are 21 kinesin motor-domain homologues and two dynein heavy chain subunits encoded in the C. elegans genome (Wormbase). We recorded time-lapse sequences of meiotic spindle movements in worms expressing GFP-tubulin and that were treated with double-stranded RNA corresponding to seven different kinesin motor-domain homologues (UNC-116/R05D3.7, KLP-3/T09A5.2, KLP-7/K11D9.1, BMK-1/F23B12.8, KLP-15/M01E11.6, KLP-18/C06G3.2, and KLP-20/Y50D7A.6). Defective meiotic spindle translocation was observed only in unc-116(RNAi) worms. UNC-116 is the C. elegans orthologue of kinesin-1 heavy chain (Patel et al., 1993; Lawrence et al., 2004). Maturing C. elegans oocytes move into a somatic structure called the spermatheca after germinal vesicle breakdown, and then squeeze out of the other side of the spermatheca into the uterus. In the examples shown in Fig. 1 (A and B), the wild-type spindle contacted the cortex 42 s after exit from the spermatheca, whereas the unc-116(RNAi) spindle contacted the cortex 8.2 min after exit from the spermatheca. Similar results were obtained from 27/27 time-lapse sequences of wild-type worms and 15/19 time-lapse sequences of unc-116(RNAi) worms (Table I).


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)

unc-116(RNAi) spindles are stationary during the period of meiosis when wild-type spindles translocate to the cortex. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from a wild-type worm (A) and an unc-116(RNAi) worm (B). The cell cortex was highlighted in each image and drawings corresponding to each image are included for clarity. (A) The wild-type meiosis I spindle translocated to the cortex from 0 to 0.7 min and adopted an orientation parallel to the cortex until spindle rotation from 8.7 to 9.7 min. (B) In contrast, the meiosis I spindle in the unc-116(RNAi) embryo remained stationary at a position several micrometers from the cortex from 0 to 6.0 min. Spindle shortening initiated just before the start of movement to the cortex (6.0 to 7.2 min) in the unc-116(RNAi) embryo, in contrast with the wild-type spindle, which initiated shortening long after cortical contact. Time 0 indicates exit of the zygote from the spermatheca into the uterus. Corresponding Videos 1 and 2 can be found in the online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1. (C–F) The shortest distance from the edge of the spindle to the cortex (▴) and the pole–pole spindle length (○) was measured in each frame of one representative GFP-tubulin time-lapse sequence from embryos of the indicated genotype. Time 0 is germinal vesicle breakdown. The vertical dotted line indicates the time the embryo exited the spermatheca into the uterus. (C) In wild-type embryos, spindle movement to the cortex initiated before exit from the spermatheca, and the 7.5-μm-long metaphase spindle remained at the cortex for 10 min before initiating its shortening phase. (D) In unc-116(RNAi) embryos, the meiotic spindle did not initiate movement toward the cortex until the time that spindle shortening initiated. (E) In embryos arrested at metaphase I due to a temperature-sensitive APC mutant, mat-2(ts), spindle shortening did not initiate but translocation to the cortex was normal. Thus, wild-type translocation is APC independent. (F) In mat-2(ts); unc-116(RNAi) double mutant embryos, the spindle did not shorten and never translocated to the cortex. Thus, the movement of unc-116(RNAi) spindles toward the cortex is APC dependent. (G and H) Fixed time point images of GFP-tubulin–labeled spindles in metaphase-arrested mat-2(ts) (G) or mat-2(ts); unc-116(RNAi) (H) worms. Bars, 10 μm.
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fig1: unc-116(RNAi) spindles are stationary during the period of meiosis when wild-type spindles translocate to the cortex. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from a wild-type worm (A) and an unc-116(RNAi) worm (B). The cell cortex was highlighted in each image and drawings corresponding to each image are included for clarity. (A) The wild-type meiosis I spindle translocated to the cortex from 0 to 0.7 min and adopted an orientation parallel to the cortex until spindle rotation from 8.7 to 9.7 min. (B) In contrast, the meiosis I spindle in the unc-116(RNAi) embryo remained stationary at a position several micrometers from the cortex from 0 to 6.0 min. Spindle shortening initiated just before the start of movement to the cortex (6.0 to 7.2 min) in the unc-116(RNAi) embryo, in contrast with the wild-type spindle, which initiated shortening long after cortical contact. Time 0 indicates exit of the zygote from the spermatheca into the uterus. Corresponding Videos 1 and 2 can be found in the online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1. (C–F) The shortest distance from the edge of the spindle to the cortex (▴) and the pole–pole spindle length (○) was measured in each frame of one representative GFP-tubulin time-lapse sequence from embryos of the indicated genotype. Time 0 is germinal vesicle breakdown. The vertical dotted line indicates the time the embryo exited the spermatheca into the uterus. (C) In wild-type embryos, spindle movement to the cortex initiated before exit from the spermatheca, and the 7.5-μm-long metaphase spindle remained at the cortex for 10 min before initiating its shortening phase. (D) In unc-116(RNAi) embryos, the meiotic spindle did not initiate movement toward the cortex until the time that spindle shortening initiated. (E) In embryos arrested at metaphase I due to a temperature-sensitive APC mutant, mat-2(ts), spindle shortening did not initiate but translocation to the cortex was normal. Thus, wild-type translocation is APC independent. (F) In mat-2(ts); unc-116(RNAi) double mutant embryos, the spindle did not shorten and never translocated to the cortex. Thus, the movement of unc-116(RNAi) spindles toward the cortex is APC dependent. (G and H) Fixed time point images of GFP-tubulin–labeled spindles in metaphase-arrested mat-2(ts) (G) or mat-2(ts); unc-116(RNAi) (H) worms. Bars, 10 μm.
Mentions: To determine whether or not microtubule motor proteins are involved in the translocation of the meiotic spindle to the oocyte cortex in C. elegans, we analyzed meiotic spindle translocation in worms depleted of different motor subunits by RNAi. There are 21 kinesin motor-domain homologues and two dynein heavy chain subunits encoded in the C. elegans genome (Wormbase). We recorded time-lapse sequences of meiotic spindle movements in worms expressing GFP-tubulin and that were treated with double-stranded RNA corresponding to seven different kinesin motor-domain homologues (UNC-116/R05D3.7, KLP-3/T09A5.2, KLP-7/K11D9.1, BMK-1/F23B12.8, KLP-15/M01E11.6, KLP-18/C06G3.2, and KLP-20/Y50D7A.6). Defective meiotic spindle translocation was observed only in unc-116(RNAi) worms. UNC-116 is the C. elegans orthologue of kinesin-1 heavy chain (Patel et al., 1993; Lawrence et al., 2004). Maturing C. elegans oocytes move into a somatic structure called the spermatheca after germinal vesicle breakdown, and then squeeze out of the other side of the spermatheca into the uterus. In the examples shown in Fig. 1 (A and B), the wild-type spindle contacted the cortex 42 s after exit from the spermatheca, whereas the unc-116(RNAi) spindle contacted the cortex 8.2 min after exit from the spermatheca. Similar results were obtained from 27/27 time-lapse sequences of wild-type worms and 15/19 time-lapse sequences of unc-116(RNAi) worms (Table I).

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
Related in: MedlinePlus