Department of Molecular and Cellular Biology, University of California, Davis, California, United States of America.
Wellcome Centre for Cell Biology & Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
PLoS Genet. 2023 Dec 27;19(12):e1011090. doi: 10.1371/journal.pgen.1011090. eCollection 2023 Dec.
Oocyte meiotic spindles mediate the expulsion of ¾ of the genome into polar bodies to generate diploid zygotes in nearly all animal species. Failures in this process result in aneuploid or polyploid offspring that are typically inviable. Accurate meiotic chromosome segregation and polar body extrusion require the spindle to elongate while maintaining its structural integrity. Previous studies have implicated three hypothetical activities during this process, including microtubule crosslinking, microtubule sliding and microtubule polymerization. However, how these activities regulate spindle rigidity and elongation as well as the exact proteins involved in the activities remain unclear. We discovered that C. elegans meiotic anaphase spindle integrity is maintained through redundant microtubule crosslinking activities of the Kinesin-5 family motor BMK-1, the microtubule bundling protein SPD-1/PRC1, and the Kinesin-4 family motor, KLP-19. Using time-lapse imaging, we found that single depletion of KLP-19KIF4A, SPD-1PRC1 or BMK-1Eg5 had minimal effects on anaphase B spindle elongation velocity. In contrast, double depletion of SPD-1PRC1 and BMK-1Eg5 or double depletion of KLP-19KIF4A and BMK-1Eg5 resulted in spindles that elongated faster, bent in a myosin-dependent manner, and had a high rate of polar body extrusion errors. Bending spindles frequently extruded both sets of segregating chromosomes into two separate polar bodies. Normal anaphase B velocity was observed after double depletion of KLP-19KIF4A and SPD-1PRC1. These results suggest that KLP-19KIF4A and SPD-1PRC1 act in different pathways, each redundant with a separate BMK-1Eg5 pathway in regulating meiotic spindle elongation. Depletion of ZYG-8, a doublecortin-related microtubule binding protein, led to slower anaphase B spindle elongation. We found that ZYG-8DCLK1 acts by excluding SPD-1PRC1 from the spindle. Thus, three mechanistically distinct microtubule regulation modules, two based on crosslinking, and one based on exclusion of crosslinkers, power the mechanism that drives spindle elongation and structural integrity during anaphase B of C.elegans female meiosis.
卵母细胞减数分裂纺锤体将基因组的四分之三排出到极体中，以在几乎所有动物物种中产生二倍体受精卵。这个过程的失败会导致非整倍体或多倍体后代，这些后代通常是无法存活的。准确的减数分裂染色体分离和极体排出需要纺锤体在保持其结构完整性的同时伸长。以前的研究表明，在此过程中有三种假设的活动，包括微管交联、微管滑动和微管聚合。然而，这些活动如何调节纺锤体的刚性和伸长，以及确切涉及的蛋白质仍不清楚。我们发现，秀丽隐杆线虫减数分裂后期 B 纺锤体的完整性是通过 Kinesin-5 家族马达 BMK-1、微管束蛋白 SPD-1/PRC1 和 Kinesin-4 家族马达 KLP-19 的冗余微管交联活性来维持的。通过延时成像，我们发现，单独耗尽 KLP-19KIF4A、SPD-1PRC1 或 BMK-1Eg5 对后期 B 纺锤体伸长速度几乎没有影响。相比之下，SPD-1PRC1 和 BMK-1Eg5 的双重耗尽或 KLP-19KIF4A 和 BMK-1Eg5 的双重耗尽导致纺锤体伸长更快，以肌球蛋白依赖的方式弯曲，并且极体排出错误率很高。弯曲的纺锤体经常将两组分离的染色体挤出到两个单独的极体中。在 KLP-19KIF4A 和 SPD-1PRC1 的双重耗尽后，观察到正常的后期 B 速度。这些结果表明，KLP-19KIF4A 和 SPD-1PRC1 作用于不同的途径，每个途径与调节减数分裂纺锤体伸长的单独的 BMK-1Eg5 途径冗余。微管结合蛋白 ZYG-8 的耗尽导致后期 B 纺锤体伸长速度变慢。我们发现，双皮质相关微管结合蛋白 ZYG-8DCLK1 通过将 SPD-1PRC1 排除在纺锤体外起作用。因此，三个机制上不同的微管调节模块，两个基于交联，一个基于交联剂的排除，为驱动 C.elegans 雌性减数分裂后期 B 纺锤体伸长和结构完整性的机制提供动力。
