Institute of Cell Biology, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zurich, Switzerland.
J Cell Biol. 2010 Sep 20;190(6):991-1003. doi: 10.1083/jcb.201006059.
Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell-level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.
von Hippel-Lindau (VHL) 肿瘤抑制基因的突变使携带者易患肾癌。已经表明 pVHL 蛋白与微管 (MT) 相互作用，这对于纤毛维持和有丝分裂纺锤体定向至关重要。然而，pVHL 在调节 MT 动力学方面的功能尚不清楚。我们通过加尾标记物 EB3（末端结合蛋白 3）-GFP 来跟踪 MT 的生长，并通过对活细胞成像中生长轨迹的时空分组，间接地推断 MT 动力学的其他参数。我们的数据将 pVHL 确立为一种近乎最佳的 MT 稳定蛋白：它在 MT 生长和收缩过程中减弱微管周转率，抑制灾难，增加救援频率。这些功能部分是通过体外和 MT 加尾以及体内 MT 晶格上抑制微管鸟嘌呤核苷酸三磷酸酶活性来介导的。与 VHL 癌症综合征相关的突变体在这些活性中受到不同程度的损害。因此，对 pVHL MT 调节功能的单细胞水平分析可以对基因型与表型的关联进行新的预测，这些预测与更粗糙的临床定义的突变体分类不同。
