Filament formation of MSF-A, a mammalian septin, in human mammary epithelial cells depends on interactions with microtubules
Filament formation of MSF-A, a mammalian septin, in human mammary epithelial cells depends on interactions with microtubules
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DOI：
10.1074/jbc.m205246200
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发表时间：
2003-05-16
影响因子：
4.8
通讯作者：
Inagaki, M
中科院分区：
文献类型：
--
作者：
Nagata, K;Kawajiri, A;Inagaki, M
Septins是一个保守的蛋白质家族，参与多种细胞功能，如胞质分裂和囊泡运输，但它们的性质和作用模式在很大程度上是未知的。在这里，我们报告一种哺乳动物Septin，MSF-A的免疫细胞化学和生化特征的研究结果。利用针对MSF亚家族蛋白的抗体，发现MSF-A主要在乳腺人乳腺上皮细胞(HMEC)中表达。MSF-A定位于有丝分裂中期的纺锤体和微管束，与间期HMEC细胞中的微管相关。生化分析表明，MSF-A通过含有鸟嘌呤核苷酸相互作用基序的中心区与聚合微管蛋白直接结合。然而，这种关联并不需要GTPase活性。破坏微管网络的条件也破坏了含有MSF-A的细丝结构，导致点状细胞质模式。使用小干扰RNA耗尽MSF-A会导致细胞不完全分裂，并导致双核细胞的积累。与Nedd5不同，缺乏GTP酶活性的MSF突变体在COS细胞中形成与野生型难以区分的细丝。这些结果有力地表明，间隔素丝不仅可以与肌动蛋白细丝相互作用，还可以与微管网络相互作用，MSF-A的GTP酶活性并不是MSF-A掺入间隔素丝所必需的。
Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.