Actin polymerization generates driving force for cell edge protrusion. Single-molecule speckle microscopy is a potent tool for probing association-dissociation kinetics of actin regulators with actin in living cells. The previous study of actin speckle analysis revealed the ceaseless actin filament turnover throughout lamellipodia of cultured fibroblasts. In order to understand how this fast actin filament lifetime is regulated, we are now extending application of single-molecule imaging to major actin end binding proteins. We also describe our discovery of processive actin capping by Formin-homology proteins, and discuss recent progress made on the mechanistic property and the structural feature of Formins’ interaction with the actin filament.

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Apoptosis is a physiological/pathophysiological form of cell death. An apoptotic volume decrease, which is induced by water efflux driven by ion effluxes under apoptotic stimulation, is a pivotal event triggering a cell to undergo apoptosis. Electrophysiological experiments revealed that an inducer of mitochondrion- or death receptor-mediated apoptosis activates volume-sensitive outwardly rectifying (VSOR) Cl^- channels that are usually involved in cell volume regulation. The channel activation via a mitochondrial pathway, but not a death receptor pathway, is mediated by reactive oxygen species (ROS). Here, we describe that VSOR Cl^- channels can sense the intracellular ROS level and that ROS-induced VSOR anion channel activation is essential for mitochondrion-mediated apoptosis processes.

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More than 1,000 rhodopsins and their related pigments (rhodopsins) have been identified thus far. The counterion, a negatively-charged amino acid residue which stabilizes a proton on the retinylidene Schiff base of the chromophore in rhodopsin is crucial for receiving a visible light. The mutational analyses by using the vertebrate and invertebrate rhodopsins revealed that counterion displacement from Glu181 to Glu113 had occurred during the molecular evolution of rhodopsins. Further biochemical analyses suggested that acquiring a new counterion Glu113 could be an essential replacement for not only emergence of efficient activation of G protein but also emergence of red-sensitive visual pigments.

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The sensor of blue-light using FAD (BLUF), a novel blue-light photoreceptor that uses flavin as a chromophore, has been found to control wide variety of light-dependent physiological responses in several microorganisms. The photocycle reaction of BLUF proteins is unique, which is characterized by a 10 nm red shift of the absorption of bound flavin in the visible region spectrum. In this review, physiological functions of several BLUF proteins are summarized. Molecular mechanisms of photocycle reaction of BLUF proteins are also discussed based on the recent advances in their spectroscopic and structural analyses.

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Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cell. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely-related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at 2.3 Å resolution, and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Among the three unique structural features of human TrpRS, deletion analyses revealed that only removal of the tRNA-anticodon-binding domain insertion, consisting of 8 residues, abolished the apoptotic activity for endothelial cells, while the translational catalytic activity and cell-binding activity remained unchanged. Thus, this work identified the inserted peptide motif important to activate the angiostatic signaling.

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