Yuki Shirai

Membrane molecules mobile even after chemical fixation

Lyn, H-ras, Cbp and TfR (we observed ~70% immobilization of GPI-anchored proteins, on average, depending strongly on the protein; Fig. 1b and Supplementary Figs. 3–5). Increasing glutaraldehyde concentration to 0.2% or methanol (100%) fixation resulted in immobilization of >80% GPI-anchored proteins but not of phospholipids or cholesterol. We then investigated antibody-induced clustering under different fixation conditions by measuring the fluorescence intensities of individual spots (Supplementary Fig. 6). Whereas treatment with 4% paraformaldehyde with or without 0.1% glutaraldehyde did not itself induce clustering, paraformaldehyde treatment did not block antibody-induced clustering of Halo-GPI and Halo–H-ras. Including 0.1% glutaraldehyde in addition to 4% paraformaldehyde suppressed clustering but did not entirely inhibit it. These results indicate that, in interpreting immunolocalization data, antibody-induced clustering Membrane molecules mobile even after chemical fixation

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single‐molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano‐meso‐scales (2–200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time‐dependent inhomogeneous two‐dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.

Rac1 Recruitment to the Archipelago Structure of the Focal Adhesion through the Fluid Membrane as Revealed by Single-Molecule Analysis

The focal adhesion (FA) is an integrin‐based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress‐fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter‐island channels in the FA zone rather freely, and the integrins in the FA‐protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G‐protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively‐active Rac1 mutant exhibited temporary and all‐time immobilizations in the FA zone, suggesting that upon PIX‐induced Rac1 activation at the FA‐protein islands, Rac1 tends to be immobilized at the FA‐protein islands.

Synthesis, water adsorption, and proton conductivity of solid-solution-type metal-organic frameworks al(oh)(bdc-oh)x(bdc-nh2) 1-x

Mixed-ligand metal-organic frameworks Al(bdc-OH)(x)(bdc-NH2)(1-x) (H2bdc-NH2 = aminoterepthalic acid, H2bdc-OH = hydroxyterephthalic acid) were synthesized and their water adsorption behavior and proton conductivity were investigated. All obtained compounds were isostructural to MIL-53 (MIL = Materials of Institut Lavoisier) according to XRD measurements under ambient humidity conditions, and were also found to be single phase across the whole mixing ratio from the XRD measurements under humidified conditions. This result clearly shows that all compounds are a solid-solution-type mixture of ligands. MIL-53-NH2 adsorbs one water molecule per formula with humidification whereas MIL-53-OH adsorbs five water molecules. The mixing ratio of the ligands in Al(OH)(bdc-OH)(x)(bdc-NH2)(1-x) affected the gate-opening pressure for water adsorption and total water uptake. Proton conductivity of these compounds largely depends on the adsorbed amount of water, which indicates that the proton conductivity of these compounds depends strongly on the hydrogen-bond network of the conducting media.

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”.