Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3 beta regulation, but did not promote insulin-stimulated GLUT4 translocation or glucose transport in L6 myotubes

We have developed a simple, direct and sensitive method to detect GLUT4 on the cell surface. Using this system, we found that PI3-kinase plays a key role in the signaling pathway of insulin-stimulated GLUT4 translocation. One of the down stream effectors of PI3-kinase is serine-threonine kinase Akt (protein kinase B, RAK-PK), but the involvement of Akt in insulin-stimulated GLUT4 translocation is controversial. To investigate whether Akt1 regulates insulin-stimulated GLUT4 translocation and glucose uptake in L6 myotubes, we established L6 myotubes stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and mouse wild type (WT) Akt1. We found that overexpression of WT Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and increased the basal activity of GSK3β, but did not promote insulin-stimulated GLUT4translocation or glucose uptake. These data supported the result that Akt is not a main signaling molecule to transmit the signal of insulin-stimulated GLUT4 translocation or glucose uptake from insulin-activated PI3-kinase

Phase separation of an actin nucleator by junctional microtubules regulates epithelial function

Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier

Laser-assisted wet coating of calcium phosphate for surface-functionalization of PEEK

Calcium phosphate (CaP) coating is an effective method for surface-functionalization of bioinert materials and for production of osteoconductive implants. Recently, we developed a laser-assisted biomimetic process (LAB process) for facile and area-specific CaP coating. In this study, the LAB process was applied to chemically stable and mechanically durable poly(etheretherketone) (PEEK), which has become widely used as an orthopedic and dental implant material. The LAB process was carried out by irradiating pulsed Nd:YAG laser light (355 nm) onto a PEEK substrate that was immersed in supersaturated CaP solution. The CaP coating applicability depended on laser fluence, i.e., CaP successfully formed on PEEK surface after the LAB process at 2 W/cm(2). Further increase in laser fluence did not result in the successful formation. At the optimal fluence of 2 W/cm(2), the laser-irradiated PEEK surface was modified and heated to induce heterogeneous CaP precipitation within 10 min in CaP solution, followed by further CaP growth over the irradiation time (tested up to 30 min). The LAB process improved the cytocompatibility of PEEK surface with osteoblastic MC3T3-E1 cells. Furthermore, the LAB-processed CaP-coated PEEK substrate formed a dense hydroxyapatite layer on its surface in the simulated body fluid, suggesting the osteoconductivity of this material. The present LAB process can be a useful new tool to produce osteoconductive PEEK-based implants

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-Pi release states are unfavourable for them to remain as filaments

Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail

Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C-terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI