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Molecular mechanisms of functional differentiation and morphogenesis of cell membranes based on the positional information of membrane phospholipids

基于膜磷脂位置信息的细胞膜功能分化和形态发生的分子机制

基本信息

批准号：
15207015
负责人：
UMEDA Masato
金额：
$ 31.28万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

Although cellular morphogenesis requires coordinated movements between the cytoskeletal and membrane systems, little is known how the orchestrated changes in the cytoskeleton and membranes are achieved during cell polarization. Using the phospholipid-binding probes, we have studied the localization and function of membrane phospholipids during cytokinesis. We found that exposure of the membrane phospholipid, phosphatidylethanolamine (PE), on the surface of the cleavage furrow membrane as a result of enhanced transbilayer movement was essential for the disassembly of the actin contractile ring and subsequent completion of cytokinesis. This observation prompted us to further analyze how the change in membrane lipid distribution affects the reorganization of actin cytoskeleton. We have revealed that the exposure of PE on the outside of the cleavage furrow accelerates downregulation of small GTPase RhoA, thereby allowing the contractile ring disassembly and completion of cytokinesis. This … More PE movement is also linked to the local production of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P_2] in the inside of cleavage furrow, which is shown to play a crucial role in completion of cytokinesis. Over-expression of kinase-deficient mutant of PI(4)P5-kinase blocked cytokinesis by preventing local accumulation of PI(4,5)P_2 at the cleavage furrow. Furthermore, microinjection of anti- PI(4,5)P_2 monoclonal antibodies blocked cytokinesis. These results suggest that the localized changes in lipid asymmetry and composition resulted in the formation of a unique lipid domain at the cleavage furrow. This lipid domain may play a role in recruiting the functional molecules that are involved in orchestrating the cytoskeletal and membrane systems to achieve successful cell division.To identify the molecules involved in the orchestrated changes in the cytoskeleton and membrane lipids, we have established budding yeast mutants that have a defect in the transbilayer movement of phospholipids. A membrane protein, designated as Ros3p, was identified as a regulator of transbilayer movement of PE across the yeast plasma membrane. Ros3p deficient cells exhibited abnormal morphology and disorganized cortical actin patches. Overproduction of Ros3p caused multibudded cells. These results suggest that Ros3p is involved in both the regulation of phospholipid movement and the actin organization in yeast. Ros3p is highly conserved in various organisms from yeast to mammals. To investigate its cellular functions, we have cloned a mammalian homolog of Ros3p, referred to as mROS3. Like in yeast, knockdown of mROS3 expression by small interfering RNAs (siRNA) was defective in inward movement of fluorescence-labeled analogs of phosphatidylserine (PS) across the plasma membrane in CHO cells. Moreover, mouse P-type ATPase colocalized with mROS3 at the microtubule organizing center and perinuclear region. In mROS3 knockdown cells, P-type ATPase could not exit from ER, implying that the decreased uptake of PS resulted from mislocalization of P-type ATPase. These results suggest that one of the cellular functions of mROS3 is serving as an escort protein that is responsible for the proper localization of P-type ATPase in mammalian cells. Further analyses of biological function of mROS3 using mROS3-knockdown and-overproducing cell lines and Drosophila mutants revealed that ROS3 protein plays a critical role in controlling the size and locomotive activity of the cells. Less

尽管细胞形态发生需要细胞骨架和膜系统之间的协调运动，但人们对细胞极化过程中如何实现细胞骨架和膜的精心安排的变化知之甚少。使用磷脂结合探针，我们研究了胞质分裂过程中膜磷脂的定位和功能。我们发现，由于跨双层运动增强，膜磷脂磷脂酰乙醇胺（PE）暴露在卵裂沟膜表面，这对于肌动蛋白收缩环的分解和随后胞质分裂的完成至关重要。这一观察结果促使我们进一步分析膜脂分布的变化如何影响肌动蛋白细胞骨架的重组。我们发现，PE 暴露在卵裂沟外侧会加速小 GTP 酶 RhoA 的下调，从而允许收缩环解体和完成胞质分裂。这种 PE 运动还与卵裂沟内部磷脂酰肌醇 4,5-二磷酸 [PI(4,5)P_2] 的局部产生有关，这在胞质分裂的完成中发挥着至关重要的作用。 PI(4)P5 激酶的激酶缺陷突变体的过度表达通过阻止 PI(4,5)P_2 在卵裂沟处的局部积累来阻断胞质分裂。此外，显微注射抗PI(4,5)P_2单克隆抗体可阻断胞质分裂。这些结果表明，脂质不对称性和组成的局部变化导致在卵裂沟处形成独特的脂质结构域。该脂质结构域可能在招募参与协调细胞骨架和膜系统以实现成功细胞分裂的功能分子方面发挥作用。为了识别参与细胞骨架和膜脂质协调变化的分子，我们建立了在磷脂跨双层运动中存在缺陷的芽殖酵母突变体。一种被命名为 Ros3p 的膜蛋白被确定为 PE 跨酵母质膜跨双层运动的调节剂。 Ros3p 缺陷细胞表现出异常的形态和紊乱的皮质肌动蛋白斑块。 Ros3p 的过量产生导致了多芽细胞。这些结果表明，Ros3p 参与酵母中磷脂运动和肌动蛋白组织的调节。 Ros3p 在从酵母到哺乳动物的各种生物体中高度保守。为了研究其细胞功能，我们克隆了 Ros3p 的哺乳动物同源物，称为 mROS3。与酵母一样，通过小干扰 RNA (siRNA) 敲低 mROS3 表达，在 CHO 细胞中荧光标记的磷脂酰丝氨酸 (PS) 类似物穿过质膜的向内运动方面存在缺陷。此外，小鼠 P 型 ATP 酶与 mROS3 共定位于微管组织中心和核周区域。在 mROS3 敲低细胞中，P 型 ATP 酶无法从 ER 中退出，这表明 PS 摄取减少是由于 P 型 ATP 酶定位错误造成的。这些结果表明，mROS3 的细胞功能之一是充当护航蛋白，负责 P 型 ATP 酶在哺乳动物细胞中的正确定位。使用 mROS3 敲低和过量产生的细胞系和果蝇突变体对 mROS3 的生物学功能进行进一步分析，结果表明 ROS3 蛋白在控制细胞的大小和运动活性方面发挥着关键作用。较少的