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

虽然细胞形态发生需要细胞骨架和膜系统之间的协调运动，但很少有人知道细胞骨架和膜的协调变化是如何在细胞极化过程中实现的。利用磷脂结合探针，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，暴露的膜磷脂，磷脂酰乙醇胺（PE），作为增强transbilayer运动的结果的分裂沟膜的表面上是必不可少的肌动蛋白收缩环的拆卸和随后完成的胞质分裂。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。我们已经发现，PE的卵裂沟外的曝光加速下调小GTdR的RhoA，从而使收缩环解体和胞质分裂的完成。这 ...更多信息 PE运动还与卵裂沟内磷脂酰肌醇4，5-二磷酸[PI（4，5）P_2]的产生有关，PI（4，5）P_2在胞质分裂的完成中起着重要作用。PI（4）P5-激酶的激酶缺陷突变体的过量表达通过阻止PI（4，5）P_2在卵裂沟的局部积累而阻断胞质分裂。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形成一个独特的脂质结构域。该脂质结构域可能在募集参与协调细胞骨架和膜系统以实现成功的细胞division.To识别参与协调细胞骨架和膜脂质变化的分子中发挥作用，我们已经建立了芽殖酵母突变体，其在磷脂的跨双层运动中具有缺陷。一种膜蛋白，命名为Ros 3 p，被确定为PE跨酵母质膜的跨双层运动的调节剂。Ros 3 p缺陷的细胞表现出异常的形态和混乱的皮质肌动蛋白补丁。Ros 3 p的过量产生导致多芽细胞。这些结果表明，Ros 3 p参与了酵母中磷脂运动和肌动蛋白组织的调节。Ros 3 p在从酵母到哺乳动物的各种生物中高度保守。为了研究其细胞功能，我们克隆了Ros 3 p的哺乳动物同源物，称为mROS 3。像在酵母中一样，通过小干扰RNA（siRNA）敲低mROS 3表达在CHO细胞中磷脂酰丝氨酸（PS）的荧光标记类似物穿过质膜的向内运动中是有缺陷的。此外，小鼠P型ATP酶与mROS 3共定位于微管组织中心和核周区。在mROS 3基因敲除的细胞中，P型ATP酶不能从内质网中排出，这意味着PS摄取的减少是由于P型ATP酶的错误定位所致。这些结果表明，mROS 3的细胞功能之一是作为一个护送蛋白，负责在哺乳动物细胞中的P型ATP酶的适当定位。使用mROS 3敲低和过量产生的细胞系和果蝇突变体对mROS 3的生物学功能的进一步分析显示，ROS 3蛋白在控制细胞的大小和运动活性中起关键作用。少

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