Department of Biology, Brooklyn College of the City University of New York, Brooklyn, New York, USA.
Cytoskeleton (Hoboken). 2011 Apr;68(4):220-36. doi: 10.1002/cm.20506.
Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the macronucleus is accompanied by elongation of an intramacronuclear microtubule array, which is oriented parallel to the axis of nuclear elongation. Elongation of the macronucleus often fails to occur or is only partially completed in a MYO1 knockout, and division of the macronucleus is frequently uncoupled from cytokinesis. Myo1 contains a myosin tail homology 4 (MyTH4) and a band 4.1, ezrin, radixin, moesin homology (FERM) domain. Recently, we used green fluorescent protein (GFP) fusions to demonstrate that the entire FERM domain, independent of MyTH4, is essential for localization of FERM to the cytoskeleton and does not appear to directly affect nuclear division. Antiactin coprecipitates GFP-FERM, tubulin, actin, and Myo1. The immunoprecipitated GFP-FERM cosediments with either exogenous F-actin or exogenous microtubules. Here, we show that overexpressed GFP-MyTH4 colocalized with antitubulin to intramacronuclear microtubules. Ninety percent of overexpressing cells assembled intramacronuclear microtubules that did not become organized into a parallel array. Amitosis did not advance in the absence of the parallel array of intramacronuclear microtubules. Five percent of overexpressing cells organized the parallel array, but the microtubules and the macronucleus did not achieve full elongation. Partially elongated macronuclei constricted without cytokinesis. Antiactin coprecipitated GFP-MyTH4, tubulin, and actin. AntiGFP pulled down GFP-MyTH4, tubulin, and actin. GFP-MyTH4 cosedimented with either exogenous microtubules or exogenous F-actin. A novel finding from this study is that MyTH4 and FERM have overlapping and distinct roles in the function of a myosin.
Myo1 是一种 XIV 类四膜虫肌球蛋白，参与无丝分裂过程中麦克伦核的伸长和收缩，将其分为两个亚核。麦克伦核的伸长伴随着内部微管阵列的伸长，该微管阵列与核伸长的轴平行排列。在 MYO1 敲除中，麦克伦核的伸长通常无法发生或仅部分完成，而且麦克伦核的分裂经常与胞质分裂脱耦。Myo1 含有肌球蛋白尾同源 4（MyTH4）和带 4.1、ezrin、radixin、moesin 同源（FERM）结构域。最近，我们使用绿色荧光蛋白（GFP）融合来证明，FERM 结构域的整个部分，独立于 MyTH4，对于 FERM 定位到细胞骨架是必不可少的，并且似乎不会直接影响核分裂。抗肌动蛋白共沉淀 GFP-FERM、微管蛋白、肌动蛋白和 Myo1。免疫沉淀的 GFP-FERM 与外源性 F-肌动蛋白或外源性微管共沉淀。在这里，我们表明，过表达的 GFP-MyTH4 与抗微管蛋白共定位到麦克伦核内部微管上。90%的过表达细胞组装了麦克伦核内部微管，但这些微管没有组织成平行阵列。在没有平行排列的麦克伦核内部微管的情况下，无丝分裂无法前进。5%的过表达细胞组织了平行排列，但微管和麦克伦核没有完全伸长。部分伸长的麦克伦核在没有胞质分裂的情况下收缩。抗肌动蛋白共沉淀 GFP-MyTH4、微管蛋白和肌动蛋白。抗 GFP 下拉 GFP-MyTH4、微管蛋白和肌动蛋白。GFP-MyTH4 与外源性微管或外源性 F-肌动蛋白共沉淀。本研究的一个新发现是，MyTH4 和 FERM 在肌球蛋白的功能中具有重叠和不同的作用。
