Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
J Cell Biol. 2010 Jan 11;188(1):49-68. doi: 10.1083/jcb.200908150.
We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator.
我们通过对果蝇胚胎的体外和体内数据进行建模，检验了星体期、前中期双极有丝分裂纺锤体由作用于纺锤体两极的外向力和内向力平衡维持的经典假说，该假说认为驱动蛋白-5 和 -14 可以对纺锤体施加向外和向内的力。在整个前中期，两种马达的斑点都排列在极间微管（ipMTs）上，马达的扰动改变了纺锤体的长度，这与预测的结果一致。纯化的驱动蛋白-5 和 -14 的竞争运动可以很好地用一个包含马达动力学和负载相关脱离的随机、相反动力冲程模型来描述。该模型的马达参数被应用于一个新的前中期纺锤体的随机力平衡模型，与胚胎的数据拟合良好。虚拟纺锤体的维持需要动态的 ipMTs 和与胚胎中发现的匹配的驱动蛋白-5 到驱动蛋白-14 的比值范围。功能扰动和建模表明，通过分解围绕前中期纺锤体的层粘连蛋白 B 包膜，可以显著扩展这个范围，从而增强精细调节的、拮抗的驱动蛋白力平衡，以在微管组装和染色体被推向赤道时维持稳定的前中期纺锤体。
