Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells.
Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells.
复制标题
半透明的哺乳动物同源物 mDia1 定位于 HeLa 细胞中的有丝分裂纺锤体。
作者：
Takayuki Kato;N. Watanabe;Yosuke Morishima;A. Fujita;T. Ishizaki;S. Narumiya
mDia 1是透明果蝇的哺乳动物同源物，并且作为小GTdR Rho的效应物起作用。它是Rho同源（FH）蛋白的成员，并且在其N末端含有Rho结合结构域和FH 3区，在中间含有聚脯氨酸延伸的FH 1区和在C末端的FH 2区。多项证据表明mDia 1和透明蛋白在胞质分裂中至关重要。mDia 1大量存在于间期细胞和有丝分裂细胞的细胞质中。使用瞬时固定的方法，优先提取可溶性成分，我们分析了本地化的mDia 1在有丝分裂的HeLa细胞。使用多克隆抗-mDia 1抗体的免疫细胞化学显示定位于有丝分裂纺锤体的特异性免疫荧光。这种定位从前期到末期都可以看到。Western印迹分析还检测到抗mDia 1免疫反应性的有丝分裂纺锤体部分分离有丝分裂HeLa细胞。与此一致，全长mDia 1作为与绿色荧光蛋白（GFP）的融合蛋白的表达在HeLa细胞的有丝分裂纺锤体中再次显示GFP荧光。mDia 1的各种截短突变体的GFP融合物的表达鉴定了这种定位是由Rho结合结构域和FH 1区域之间的173个氨基酸长的序列决定的，所述FH 1区域包含FH 3区域的C末端部分。点突变分析表明，FH 3区的Leu（434）和Leu（455）对于有丝分裂纺锤体的定位至关重要。无论是电穿孔的肉毒杆菌C3外切酶，也不微量注射Val 14 RhoA到有丝分裂细胞的影响本地化的内源性mDia 1的有丝分裂纺锤体，这表明mDia 1本地化的有丝分裂纺锤体独立的Rho活性。因此，本研究建立了mDia 1定位在有丝分裂纺锤体。这种定位表明mDia 1在细胞分裂过程中纺锤体-卵裂沟相互作用中的作用。
mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.
影响因子：
3.3
作者：
Cao, LG;Wang, YL
通讯作者：
Wang, YL