I propose to call the cellular mechanism by which membrane molecules are organized to form oligomers, assemblies, or localized structures as “membrane organizers.” For the understanding of membrane functions, the knowledge on the mechanism by which relevant molecules are structurally arranged and organized in the membrane is essential. Membrane research from the standpoint of “membrane organizers” is emphasized.
Lateral diffusion coefficient of transmembrane proteins in the plasma membrane is smaller than that in artificial membranes by a factor of 1/10-1/100. We studied the restriction mechanism of the lateral mobility of membrane proteins in the plasma membrane by using single particle tracking technique and optical tweezers. These techniques revealed that the movement of many membrane proteins is regulated from the cytoplasmic side through the interaction with the network of the membrane-skeleton/cytoskeleton. Two major ways of such interaction are corralling effect of the fence of the membrane skeleton and tethering to the cytoskeleton.
To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, or tubulin and microtubule associated proteins (MAPs), using high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-action polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape. This shape was stabilized only when MAPs were co-encapsulated.
Recent advances in molecular genetics have provided powerful tools for studying the biological function of proteins, resulting in rapid extension of our knowledge of their functions. However, little is still known about the organization and function of biological membranes, especially how membrane phospholipids are organized and participate in cellular functions. This is mainly because of the lack of appropriate methodologies for either manipulating or tracing the function of membrane lipids. We have developed various probes to analyze the molecular motion and function of phospholipids in biological membranes. Our recent studies have shown that enhanced transbilayer movement of plasma membrane phosphatidylethanolamine plays a pivotal role in mediating coordinated movement between cytoskeletal proteins and plasma membrane to achieve successful cell division and membrane fusion.
Influenza virus hemagglutinin is a paradigm for biological membrane fusion mechanisms. Among the most important recent progresses in this field are “spring-loaded mechanism” based on a coiled-coil of α-helices and “microprotrusion of lipid bilayer” which was found through a quick-freezing electron microscopic observation. Possible molecular architectures of membrane fusion intermediates induced by hemagglutinin are discussed.
In mammalian cells, especially in polarized cells, the Golgi apparatus plays a crucial role in the instracellular sorting and directional-transport of proteins and lipids. The dynamic nature of the apparatus is underscored by the cell cycle-dependent redistribustion and coalesce near the restricted pericentriolar and apical locarlzation in polarized cells. Cytoskeletons might be involved in the dynamic nature, the characteristic shape and the intracellular position of the Golgi apparatus. However, the precise mechanisms of the Golgi-positioning and of the maintenance of its shape in polarized cells have not been clarified yet. To address this problem, we have developed “semi-intact cell” system to study the molecular mechanisms of the “topo-biogenesis” of the Golgi apparatus in polarized MDCK cells.
Autophagy is ubiquitous and fundamental cellular activity in every eukaryotic cell. It is a process for the bulk degradation of cytoplasmic proteins and excess organelles, in which a portion of cytoplasm are enclosed to form double membrane structures known as autophagosomes for delivery to lysosome/vacuole for degradation. This process is necessary for survival under starvation and cell differentiation. So far molecular basis of autophagy is poorly understood. We are studying autophagy using yeast, Saccharomyces cerevisiae, as a model system. We have identified 14 genes essential for autophagy in the yeast and found that most of these APG genes are novel. Recently among these genes new protein-conjugation system necessary for autophagy was found. Further analyses on these genes will uncover the mechanism of autophagy at a molecular level.