Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization
Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization
复制标题
Septin 7 与着丝粒相关蛋白 E 相互作用，是其着丝粒定位所必需的
DOI：
10.1074/jbc.m710591200
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发表时间：
2008-07-04
影响因子：
4.8
通讯作者：
Yao, Xuebiao
中科院分区：
文献类型：
--
作者：
Zhu, Mei;Wang, Fengsong;Yao, Xuebiao
有丝分裂中染色体的分离是由纺锤体微管和动粒之间的动态相互作用协调的。Septin（SEPT）是一个保守的GTP酶家族，定位于有丝分裂中期纺锤体。先前的研究表明，SEPT 2缺失导致染色体错误分离，这与聚集染色体的着丝粒相关蛋白E（CENP-E）丢失有关（1）。然而，CENP-E是否与SEPT物理相互作用以及这种相互作用如何在有丝分裂中协调染色体分离仍然是难以捉摸的。在这里，我们表明，SEPT 7是需要一个稳定的着丝粒定位CENP-E在HeLa和MDCK细胞。SEPT 7通过与CENP-E的C-末端结构域直接相互作用来稳定CENP-E的动粒缔合。通过谷胱甘肽S-转移酶下拉和酵母双杂交测定将SEPT 7与CENP-E结合的区域定位到其C-末端结构域。免疫荧光研究显示SEPT 7纤维沿着有丝分裂纺锤体分布，终止于CENP-E标记的动粒。值得注意的是，通过小干扰RNA抑制SEPT 7的合成，消除了CENP-E在动粒上的定位，并引起异常的染色体分离。CENP-E的这些有丝分裂缺陷和动粒定位可以通过将外源GFP-SEPT 7引入SEPT 7耗尽的细胞中成功地挽救。这些SEPT 7抑制的细胞在双向染色体的动粒处显示出降低的张力，并且在这些未对齐的染色体上显示出由Mad 2和BubR 1标记标记的激活的有丝分裂纺锤体检查点。这些发现揭示了SEPT 7-CENP-E相互作用在CENP-E分布到动粒和实现染色体比对中的关键作用。
Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E ( CENP-E) from the kinetochores of congressing chromosomes ( 1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP- E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP- E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP- E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi- orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment.