Hogan C J, Stephens L, Shimizu T, Cande W Z
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Cell Biol. 1992 Dec;119(5):1277-86. doi: 10.1083/jcb.119.5.1277.
We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.
我们开发了一种新的体外研究纺锤体伸长的模型系统，使用的是羽状海洋硅藻梭形筒柱藻。梭形筒柱藻在对数生长期可大量培养至高细胞密度，并通过简单的光/暗周期实现同步化。可以获得足够数量用于生化分析的分离纺锤体，纺锤体微管蛋白约占总蛋白量的5%。纺锤体分离程序使硅藻微管蛋白富集了10倍，纺锤体蛋白计算增加了40倍。分离的纺锤体或通透细胞中的纺锤体在体外能够以与其他后期B模型（Cande和McDonald，1986；Masuda等人，1990）所描述的相同机制和相同药理学敏感性进行伸长。利用该模型，针对一系列三磷酸核苷酸和ATP类似物绘制了体外纺锤体伸长速率曲线。各种三磷酸核苷酸产生的纺锤体伸长相对速率与微管滑动试验中基于驱动蛋白的运动所观察到的相对速率平行。同样，能够区分肌球蛋白、动力蛋白和驱动蛋白介导运动的ATP类似物产生了驱动蛋白运动特有的纺锤体伸长相对速率。此外，通过针对驱动蛋白超家族保守区域的肽抗体鉴定，分离的纺锤体组分富含一种与驱动蛋白相关的蛋白质。这些数据表明，类似驱动蛋白的运动在有丝分裂后期B期间对纺锤体伸长有贡献。
