Limits...
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.

Show MeSH

Related in: MedlinePlus

Dynein heavy chain is not required for preanaphase spindle translocation. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from dhc-1(RNAi) worms. The cell cortex was highlighted in each image for clarity. (A) Worms observed at short time points after soaking in dhc-1 dsRNA exhibited bipolar meiotic spindles that translocated to the cortex immediately after exit from the spermatheca (asterisk), long before initiation of spindle shortening. Two female pronuclei formed at the end of this sequence and these pronuclei did not migrate toward the male pronucleus as reported by Gonczy et al. (1999). (B) Worms observed at longer time points after soaking in dhc-1 dsRNA had multiple germinal vesicles in the diakinesis-stage oocytes before maturation. The 0 min image shows an oocyte during germinal vesicle breakdown as GFP-tubulin is polymerizing within each fenestrated nucleus. All of these spindles ended up at the cortex (30.5 min) after coalescing into a smaller number of spindles. (C) Fixed time point image of a mat-2(ts); dhc-1(RNAi) worm shows a disorganized spindle that is tightly associated with the cortex. (D) Maximum intensity projection of a z-stack of spinning disk confocal images of GFP-tubulin fluorescence in a living, wild-type worm. Brightness has been adjusted to reveal the cytoplasmic microtubule array in the meiotic embryo on the left and the immature oocyte on the right. The black region in between is the spermatheca. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171918&req=5

fig6: Dynein heavy chain is not required for preanaphase spindle translocation. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from dhc-1(RNAi) worms. The cell cortex was highlighted in each image for clarity. (A) Worms observed at short time points after soaking in dhc-1 dsRNA exhibited bipolar meiotic spindles that translocated to the cortex immediately after exit from the spermatheca (asterisk), long before initiation of spindle shortening. Two female pronuclei formed at the end of this sequence and these pronuclei did not migrate toward the male pronucleus as reported by Gonczy et al. (1999). (B) Worms observed at longer time points after soaking in dhc-1 dsRNA had multiple germinal vesicles in the diakinesis-stage oocytes before maturation. The 0 min image shows an oocyte during germinal vesicle breakdown as GFP-tubulin is polymerizing within each fenestrated nucleus. All of these spindles ended up at the cortex (30.5 min) after coalescing into a smaller number of spindles. (C) Fixed time point image of a mat-2(ts); dhc-1(RNAi) worm shows a disorganized spindle that is tightly associated with the cortex. (D) Maximum intensity projection of a z-stack of spinning disk confocal images of GFP-tubulin fluorescence in a living, wild-type worm. Brightness has been adjusted to reveal the cytoplasmic microtubule array in the meiotic embryo on the left and the immature oocyte on the right. The black region in between is the spermatheca. Bars, 10 μm.

Mentions: One hypothesis consistent with our results is that kinesin-1 transports the spindle on a subset of cytoplasmic microtubules that have plus ends oriented toward the cortex. An alternative model, suggested by work in Aspergillus nidulans (Zhang et al., 2003) and D. melanogaster (Brendza et al., 2002), is that UNC-116 is only required to localize the minus end–directed motor, cytoplasmic dynein. In this model, cytoplasmic dynein would be required to move the spindle on a subset of cytoplasmic microtubules that have minus ends oriented toward the cortex. Therefore, we tested if early meiotic spindle translocation is blocked when cytoplasmic dynein heavy chain, DHC-1, is depleted by RNAi. As previously reported (Gonczy et al., 1999), the strongest phenotype observed after prolonged treatment with dhc-1 dsRNA was failure to ovulate mature oocytes. Shorter treatment of worms with dhc-1(RNAi) resulted in meiosis I spindles with a variety of structural defects. Some spindles were longer than wild-type spindles and had extremely pointed poles, whereas others were extremely disorganized. Time-lapse imaging of GFP-tubulin–labeled spindles under these weak dhc-1(RNAi) conditions revealed that 7/7 spindles arrived at the cortex either before or within 1.0 ± 0.4 min (1.0 ± 0.7 min in wild type) after exit from spermatheca (Fig. 6 A). At longer time points of dhc-1 dsRNA treatment, before worms ceased to produce mature oocytes, multiple small diakinesis nuclei were observed in immature oocytes (Fig. 6 B). Time-lapse imaging of GFP-tubulin revealed that each of these nuclei gave rise to a spindle-like structure at germinal vesicle breakdown and that all of these structures were eventually associated with the cortex (Fig. 6 B, 30.5 min). Tracking the early movements of these spindles to determine the time of cortical contact, however, proved difficult and was not pursued. To determine whether or not the early, APC-independent translocation mechanism was functional in these dhc-1(RNAi) embryos, mat-2(ts), GFP-tubulin worms, were treated with dhc-1 dsRNA at 25°C so that metaphase-arrested meiotic embryos that were derived from oocytes with multiple germinal vesicles could be observed. The majority of meiotic spindles in these mat-2(ts); dhc-1(RNAi) double mutant embryos were associated with the cortex just as in mat-2(ts) single mutants (Fig. 6 C). These results provide no indication of a role for cytoplasmic dynein in the early, APC-independent spindle translocation. Thus, UNC-116 appears to play a more direct role in early spindle translocation, possibly by transporting the spindle on cytoplasmic microtubules that are oriented with their plus ends toward the cortex. An obvious challenge is elucidating how the acentrosomal cytoplasmic microtubule array shown in Fig. 6 D can be organized to allow directional transport.


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)

Dynein heavy chain is not required for preanaphase spindle translocation. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from dhc-1(RNAi) worms. The cell cortex was highlighted in each image for clarity. (A) Worms observed at short time points after soaking in dhc-1 dsRNA exhibited bipolar meiotic spindles that translocated to the cortex immediately after exit from the spermatheca (asterisk), long before initiation of spindle shortening. Two female pronuclei formed at the end of this sequence and these pronuclei did not migrate toward the male pronucleus as reported by Gonczy et al. (1999). (B) Worms observed at longer time points after soaking in dhc-1 dsRNA had multiple germinal vesicles in the diakinesis-stage oocytes before maturation. The 0 min image shows an oocyte during germinal vesicle breakdown as GFP-tubulin is polymerizing within each fenestrated nucleus. All of these spindles ended up at the cortex (30.5 min) after coalescing into a smaller number of spindles. (C) Fixed time point image of a mat-2(ts); dhc-1(RNAi) worm shows a disorganized spindle that is tightly associated with the cortex. (D) Maximum intensity projection of a z-stack of spinning disk confocal images of GFP-tubulin fluorescence in a living, wild-type worm. Brightness has been adjusted to reveal the cytoplasmic microtubule array in the meiotic embryo on the left and the immature oocyte on the right. The black region in between is the spermatheca. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171918&req=5

fig6: Dynein heavy chain is not required for preanaphase spindle translocation. Images of GFP-tubulin fluorescence within a meiotic embryo are shown from representative time-lapse sequences from dhc-1(RNAi) worms. The cell cortex was highlighted in each image for clarity. (A) Worms observed at short time points after soaking in dhc-1 dsRNA exhibited bipolar meiotic spindles that translocated to the cortex immediately after exit from the spermatheca (asterisk), long before initiation of spindle shortening. Two female pronuclei formed at the end of this sequence and these pronuclei did not migrate toward the male pronucleus as reported by Gonczy et al. (1999). (B) Worms observed at longer time points after soaking in dhc-1 dsRNA had multiple germinal vesicles in the diakinesis-stage oocytes before maturation. The 0 min image shows an oocyte during germinal vesicle breakdown as GFP-tubulin is polymerizing within each fenestrated nucleus. All of these spindles ended up at the cortex (30.5 min) after coalescing into a smaller number of spindles. (C) Fixed time point image of a mat-2(ts); dhc-1(RNAi) worm shows a disorganized spindle that is tightly associated with the cortex. (D) Maximum intensity projection of a z-stack of spinning disk confocal images of GFP-tubulin fluorescence in a living, wild-type worm. Brightness has been adjusted to reveal the cytoplasmic microtubule array in the meiotic embryo on the left and the immature oocyte on the right. The black region in between is the spermatheca. Bars, 10 μm.
Mentions: One hypothesis consistent with our results is that kinesin-1 transports the spindle on a subset of cytoplasmic microtubules that have plus ends oriented toward the cortex. An alternative model, suggested by work in Aspergillus nidulans (Zhang et al., 2003) and D. melanogaster (Brendza et al., 2002), is that UNC-116 is only required to localize the minus end–directed motor, cytoplasmic dynein. In this model, cytoplasmic dynein would be required to move the spindle on a subset of cytoplasmic microtubules that have minus ends oriented toward the cortex. Therefore, we tested if early meiotic spindle translocation is blocked when cytoplasmic dynein heavy chain, DHC-1, is depleted by RNAi. As previously reported (Gonczy et al., 1999), the strongest phenotype observed after prolonged treatment with dhc-1 dsRNA was failure to ovulate mature oocytes. Shorter treatment of worms with dhc-1(RNAi) resulted in meiosis I spindles with a variety of structural defects. Some spindles were longer than wild-type spindles and had extremely pointed poles, whereas others were extremely disorganized. Time-lapse imaging of GFP-tubulin–labeled spindles under these weak dhc-1(RNAi) conditions revealed that 7/7 spindles arrived at the cortex either before or within 1.0 ± 0.4 min (1.0 ± 0.7 min in wild type) after exit from spermatheca (Fig. 6 A). At longer time points of dhc-1 dsRNA treatment, before worms ceased to produce mature oocytes, multiple small diakinesis nuclei were observed in immature oocytes (Fig. 6 B). Time-lapse imaging of GFP-tubulin revealed that each of these nuclei gave rise to a spindle-like structure at germinal vesicle breakdown and that all of these structures were eventually associated with the cortex (Fig. 6 B, 30.5 min). Tracking the early movements of these spindles to determine the time of cortical contact, however, proved difficult and was not pursued. To determine whether or not the early, APC-independent translocation mechanism was functional in these dhc-1(RNAi) embryos, mat-2(ts), GFP-tubulin worms, were treated with dhc-1 dsRNA at 25°C so that metaphase-arrested meiotic embryos that were derived from oocytes with multiple germinal vesicles could be observed. The majority of meiotic spindles in these mat-2(ts); dhc-1(RNAi) double mutant embryos were associated with the cortex just as in mat-2(ts) single mutants (Fig. 6 C). These results provide no indication of a role for cytoplasmic dynein in the early, APC-independent spindle translocation. Thus, UNC-116 appears to play a more direct role in early spindle translocation, possibly by transporting the spindle on cytoplasmic microtubules that are oriented with their plus ends toward the cortex. An obvious challenge is elucidating how the acentrosomal cytoplasmic microtubule array shown in Fig. 6 D can be organized to allow directional transport.

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