Nobuhiro Morone

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

Interaction of Rho‐kinase with myosin II at stress fibres

Rho‐kinase and myosin phosphatase cooperatively regulate the phosphorylation level of myosin light chain and are involved in the formation of stress fibres and smooth muscle contraction. Rho‐kinase has been known to be localized at stress fibres, but little is known about the mechanism of its localization. Here we identified non‐muscle myosin heavy chain IIA and IIB as the pleckstrin homology domain‐interacting molecules by affinity column chromatography. The pleckstrin homology domain of Rho‐kinase binds to myosin II directly in in vitro cosedimentation assay. The C‐terminal region of the pleckstrin homology domain was important for this interaction, and the point mutations in the pleckstrin homology domain mutant (W1170A, W1340L) resulted in a decrease in the binding. We also found that the pleckstrin homology domain, but not the pleckstrin homology domain mutant (W1170A, W1340L), was localized at stress fibres in fibroblasts. These results indicate that Rho‐kinase is localized at stress fibres through binding of the pleckstrin homology domain to myosin II.

Close Interrelationship of Sphingomyelinase and Caveolin in Triton X-100-lnsoluble Membrane Microdomains

Much attention has been paid to the roles of sphingomyelin (SM) metabolism in the regulation of various cell functions such as cell growth, differentiation and apoptosis. Sphingomyelinase (SMase) catalyzes the first step of SM-metabolizing pathways that generate the bioactive metabolite, ceramide. SMase present in membrane microdomains, raft and caveolae may be involved in the agonist-mediated events. However, it is still unclear which molecular species of SMase is stimulated by an agonist to produce ceramide in membrane microdomains, and how agonist-sensitive SMase and SM are topologically localized within the micro-domains. Here, we first show the close interaction between neutral SMase and caveolin 1 in 1% Triton X-100-insoluble fractions of plasma membranes isolated from adult rat resting liver and rapidly growing rat ascites hepatoma, AH 7974 cells. Then, we describe the connection between acid SMase and caveolin 2 in cell differentiation or apoptosis induced by all-trans retinoic acid. Finally, we discuss the possible roles for caveolins with respect to the topological distribution of SMase and SM in caveolae.