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

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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