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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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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.
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fig5: 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.

Mentions: To directly demonstrate the existence of a protein complex containing UNC-116, KLC-2, and KCA-1, all three proteins were expressed as fusion proteins in E. coli and interactions were analyzed by glutathione Sepharose chromatography. As shown in Fig. 5 A (lane 10), a chitin-binding domain fusion to the UNC-116 stalk-tail domains stochiometrically copurified with a glutathione S–transferase fusion to KCA-1 only in the presence of 6his-KLC-2b. This experiment demonstrated that KLC-2b can either bind simultaneously to both UNC-116 and KCA-1 or that KLC-2b induces a conformational change that increases the affinity between UNC-116 and KCA-1. Similar experiments with KCA-1 deletion derivatives (Fig. 5 B) indicated that the NH2-terminal 155 amino acids of KCA-1 contain the KLC-2 binding domain. This result implies that the COOH-terminal part of KCA-1 may bind to some spindle component to generate a bridge between UNC-116 and the meiotic spindle. KCA-1 has no homology with sunday driver or other known kinesin-associated proteins and thus represents a novel 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)

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.
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fig5: 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.
Mentions: To directly demonstrate the existence of a protein complex containing UNC-116, KLC-2, and KCA-1, all three proteins were expressed as fusion proteins in E. coli and interactions were analyzed by glutathione Sepharose chromatography. As shown in Fig. 5 A (lane 10), a chitin-binding domain fusion to the UNC-116 stalk-tail domains stochiometrically copurified with a glutathione S–transferase fusion to KCA-1 only in the presence of 6his-KLC-2b. This experiment demonstrated that KLC-2b can either bind simultaneously to both UNC-116 and KCA-1 or that KLC-2b induces a conformational change that increases the affinity between UNC-116 and KCA-1. Similar experiments with KCA-1 deletion derivatives (Fig. 5 B) indicated that the NH2-terminal 155 amino acids of KCA-1 contain the KLC-2 binding domain. This result implies that the COOH-terminal part of KCA-1 may bind to some spindle component to generate a bridge between UNC-116 and the meiotic spindle. KCA-1 has no homology with sunday driver or other known kinesin-associated proteins and thus represents a novel 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.

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