Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter - PubMed (original) (raw)
Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter
Hsin-ya Yang et al. J Cell Biol. 2005.
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.
Figures
Figure 1.
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.
Figure 2.
Polar body formation in an unc-116(RNAi) embryo. Images of GFP-histone H2b fluorescence within a meiotic embryo are shown from a representative time-lapse sequence of an unc-116(RNAi) worm. The cortex has been highlighted in each image and drawings corresponding to the top row of images are included for clarity. Note that anaphase of meiosis I is successful (18 min) but the chromosomes that should have been sequestered in a polar body snap back to produce a meiosis II spindle with 12 rather than 6 chromosomes (22.0–24.3 min). Anaphase of meiosis II was also successful (30.3 min), and the chromosomes segregated into the cortex were successfully sequestered in a polar body (36.3 min). Asterisk indicates exit of the zygote from the spermatheca into the uterus. Bar, 10 μm.
Figure 3.
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.
Figure 4.
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.
Figure 5.
In vitro reconstitution of a complex between kinesin heavy chain, kinesin light chain, and KCA-1. (A) Glutathione Sepharose beads were coated either with glutathione S-transferase (GST; lanes 1–5) or a GST fusion with aa 1–290 of KCA-1 (GST-KCA-1; lanes 6–10). Coated beads were incubated with different chitin binding domain intein fusion proteins (CBD) and washed extensively, and the bound complexes were eluted with SDS, resolved by SDS-PAGE, and detected with Coomassie brilliant blue R staining. The negative control, CBD-MEI-1, did not bind to GST (lane 1) or KCA-1 (lane 6). In contrast, both a 6his-KLC-2b fusion (lanes 2 and 7) and a CBD-KLC-2b fusion (lanes 3 and 8) bound with a high stochiometry to KCA-1 but not to the GST control. CBD-UNC-116 (lanes 4 and 9) bound to KCA-1 with a low stochiometry, but significantly more CBD-UNC-116 associated with KCA-1 beads when 6his-KLC-2b was also present (lanes 5 and 10). This result indicates that KLC-2b can form a ternary complex with both UNC-116 and KCA-1. (B) Glutathione Sepharose beads were coated with GST fusions to different deletion derivatives of KCA-1 and incubated either with the negative control, CBD-MEI-1 (lanes 1, 3, and 5), or CBD-KLC-2b (lanes 2, 4, 6, and 7). Beads were washed extensively and bound complexes were eluted with SDS, resolved by SDS-PAGE, and detected with Coomassie brilliant blue R staining. A high stochiometry of CBD-KLC-2b associated with both aa 1–415 of KCA-1 (lane 2) and aa 1–290 (lane 4). In contrast, much less CBD-KLC-2b associated with KCA-1 aa 155–415 (lane 6) or 155–290 (lane 7). These results indicate that most of the binding to KLC-2b is mediated by the NH2-terminal 155 aa of KCA-1. Note that all lanes containing CBD-KLC-2b also have a polypeptide corresponding to KLC-2b alone produced by spontaneous cleavage of the intein fusion. Also, KCA-1 derivatives that contain the COOH-terminal 125 aa migrate anomalously slowly relative to derivatives without this region.
Figure 6.
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.
Figure 7.
Model for the sequential action of the early, UNC-116–dependent spindle translocation pathway and the late, APC-dependent pathway. In wild-type worms, UNC-116 moves the spindle on cytoplasmic microtubules toward the cortex. At the APC-dependent metaphase–anaphase transition, astral microtubules extend from one pole and mediate pulling forces. In wild-type embryos, this APC-dependent cortical pulling results in rotation. In unc-116(RNAi) embryos, the same force generates translocation with one pole leading.
References
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