宮澤 淳夫

Here we present the first evidence that muscle-specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MuSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ. © 2008 New York Academy of Sciences.

The subcellular localization of biomolecules at high resolution has traditionally been investigated by combining transmission electron microscopy (TEM) and chemical staining with heavy metals or immuno-based labeling with gold-conjugated antibodies. Here, we employ genetically encoded tags to examine the localization of proteins in transfected cultured cells by TEM. We purified a fusion protein of postsynaptic density-95 (PSD-95) coupled to three tandem repeats of metallothionein (MT) (PDS-953MT) from COS7 cells grown in the presence of Cd2+. PSD-953MT was detected as black particles by TEM. To visualize the subcellular localization of PSD-953MT, expression constructs encoding this fusion protein were transfected into primary hippocampal neurons cultured in medium containing Cd2+. The subcellular accumulation of PSD-953MT and Cd2+ provided excellent contrast in TEM micrographs. To address if genetically encoded tags affect the function of the target proteins, we found that the conjugation of 3MT to PSD-95 did not alter its association with known binding partners. These results demonstrate that 3MT coordinating Cd2+ is a valuable genetically encoded tag to study the localization of proteins by TEM.

The observation of biological materials by transmission electron microscopy (TEM) frequently requires the use of negative staining andor immuno-gold labeling to visualize or identify the specimen, but these techniques are limited. In contrast, genetic labeling with green fluorescent protein (GFP) and its homologs have led to rapid advances in the observation of proteins of interest in cells by light microscopy (LM). These fluorescent tags allow for the visualization of dynamic processes in live cells without the use of secondary reagents. Here, we report the development of an artificial metalloprotein fusion protein expressed in Escherichia coli grown in Cd-2-containing medium that allows for efficient protein detection by TEM without additional staining steps. We linked the subunits of the bacterial 14-mer protein GroEL with three repeats of metallothionein (3MT). The 3MT-fused GroEL (GroEL-14(3MT)) was successfully expressed in E. coli, and the purified protein included 250 cadmium atoms per molecule on average. Cd2+-bound GroEL-14(3MT) was detected by TEM in the absence of negative staining on a carbon grid, and the particle densities of GroEL-14(3MT) were much greater than those of untagged GroEL in vitreous ice. Taken together, our data indicate that the 3MT tag provides a promising means of allowing the identification of oligomeric proteins isolated from cells in the absence of other detection techniques.

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of similar to 220 angstrom in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an similar to 600 angstrom diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin. The HOOK region of PSD-95, which is located between the src homology 3 domain and the guanylate kinase-like domain, was determined to be involved in the binding of PSD-95 to calmodulin. We also found that C-terminal peptides from proteins such as CRIPT and the N-methyl-D-aspartate receptor NR2B subunit, which associate with the PDZ domain of PSD-95, enhanced the affinity of PSD-95 for calmodulin. The binding of ligands to the PDZ domain may change the conformation of PSD-95 and affect the interaction between PSD-95 and calmodulin.

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca2+ channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca2+ signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca2+ release by co-agonists,Ca2+, inositol 1,4,5 tri-phosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain. (C) 2003 Elsevier Ltd. All rights reserved.

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.

The nicotinic acetylcholine (ACh) receptor belongs to a superfamily of synaptic ion channels that open in response to the binding of chemical transmitters. Their mechanism of activation is not known in detail, but a time-resolved electron microscopic study of the muscle-type ACh receptor had suggested that a local disturbance in the ligand-binding region and consequent rotations in the ligand-binding alpha subunits, connecting to the transmembrane portion, are involved. A more precise interpretation of this structural change is given here, based on comparison of the extracellular domain of the ACh receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. We find that, to a good approximation, there are two alternative extended conformations of the ACh receptor subunits, one characteristic of either alpha subunit before activation, and the other characteristic of all three non-alpha subunits and the protomer of AChBP. Substitution in the three-dimensional maps of a by non-a subunits mimics the changes seen on activation, suggesting that the structures of the a subunits are modified initially by their interactions with neighbouring subunits and switch to the non-alpha form when ACh binds. This structural change, which entails 15-16degrees rotations of the inner pore-facing parts of the alpha subunits, most likely acts as the trigger that opens the gate in the membrane-spanning pore. (C) 2002 Elsevier Science Ltd. All rights reserved.

PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins. (c) 2002 Elsevier Science Ltd.

Bacteriorhodopsin is a transmembrane protein that uses light energy, absorbed by its chromophore retinal, to pump protons from the cytoplasm of bacteria such as Halobacterium salinarium into the extracellular space(1,2.) It is made up of seven alpha-helices, and in the bacterium forms natural, two-dimensional crystals called purple membranes. We have analysed these crystals by electron cryo-microscopy to obtain images of bacteriorhodopsin at 3.0 Angstrom resolution. The structure covers nearly all 248 amino acids, including loops outside the membrane, and reveals the distribution of charged residues on both sides of the membrane surface. In addition, analysis of the electron-potential map produced by this method allows the determination of the charge status of these residues. On the extracellular side, four glutamate residues surround the entrance to the proton channel, whereas on the cytoplasmic side, four aspartic acids occur in a plane at the boundary of the hydrophobic-hydrophilic interface. The negative charges produced by these aspartate residues is encircled by areas of positive charge that may facilitate accumulation and lateral movement of protons on this surface.

Bacteriorhodopsin pumps protons from the cytoplasm to the outside of halobacteria, Halobacterium salinarium, by using absorbed light energy. The newly observed density map at 3 Å resolution clarified nearly the entire structure the resolution in the direction perpendicular to the membrane surface is 3.2 Å. The new structure clearly indicates the proton transfer pathway in bacteriorhodopsin. In particular, the location of key aspartic acid and glutamic acid residues in the derived structural model suggested funneling structures with different designs for input and output of protons on the cytoplasmic and extracellular sides, respectively, of the protein. This paper describes the major differences between the model based on the new observation and the former model obtained through crystallographic refinement by Grigorieff et al. (J. Mol. Biol. 259 393-421, 1996).

To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA-1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated μ-calpain demonstrated μ-calpain activation specifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry. CA-1 neurons on day 1 showed sustained activation of μ-calpain, and increased immunostaining for inactivated and activated forms of μ- and m-calpains and for PIP2. Activated μ-calpain and PIP2 were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia-hypoglycaemia in vitro provoked intense Ca2+mobilization with increased PIP2 immunostaining specifically in CA-1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca2+ and PIP2 breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA-1 neuronal death. © European Neuroscience Association.

Mobilization of [Ca2+](i) in the monkey hippocampal slices during transient hypoxia-hypoglycemia and KCl-induced deporalization was analyzed by microfluorometric imaging and anti-PIP2 immunohistochemistry. Hypoxia-hypoglycemia provoked the largest [Ca2+](i) mobilization of CA-1 temperature-dependently whereas [Ca2+](i) mobilization by KCl-induced depolarization occurred independent of the temperature in CA-2. Immunohistochemical analysis of the hippocampus after hypoxiahypoglycemia showed an increased PIP2 staining preferentially in the perikarya of CA-1 neurons. These data suggest that release of Ca2+ from intracellular stores caused by PIP2 breakdown may induce elevated [Ca2+](i). (C) 1994 Academic Press, Inc.

Abstract: Understanding the topographical relationships between phosphatidylserine (PS) and protein kinase C (PKC) within neurons can provide clues about the mechanism of translocation and activation of PKC. For this purpose we applied monoclonal antibodies (Abs) of PS and PKC to sections of developing rat cerebellum. The anti‐PKC Ab immunohistochemical pattern showed homogeneous staining of Purkinje cells over various postnatal ages, whereas the anti‐PS Ab staining showed a heterogeneous localization over these ages. Purkinje cells did not stain well between postnatal day 14 (PND 14) and PND 21, suggesting that the PS was lost from the membrane during preparation of the sections during this period. These data imply that interactions between PS and PKC vary in Purkinje cells during postnatal development. Copyright © 1992, Wiley Blackwell. All rights reserved

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N-methyl-d-aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex. © 1991.

We established a series of monoclonal antibodies (MAbs) that bound to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] which is confidently believed to play an important role in cellular signal transduction. We used complement dependent liposome lysis assay for screening MAbs and antibodies that bind to PI(4,5)P2 but not to phosphatidylinositol 4-monophosphate [PI(4)P] were selected. The reactivity of the MAbs was analyzed by complement dependent liposome lysis assay, enzyme-linked immunosorbent assay (ELISA) and inhibition of liposome lysis by soluble haptens. The MAbs exhibited three distinct reactivity profiles measured by three different assay systems and the results obtained from three typical MAbs designated AM-2, AM-7 and AM-212 are described. On liposome lysis assay, all three MAbs were highly specific with PI(4,5)P2 and no cross-reaction with other acidic phospholipids such as PI(4)P, phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE) and cardiolipin (CL) was observed. All three MAbs were able to react with as low as 0.1 mol% of PI(4,5)P2 embedded in the liposomal membrane. On ELISA, AM-2 and AM-212 bound only to PI(4,5)P2 and did not cross-react with other acidic phospholipids including PI(4)P. In contrast, AM-7 showed considerable binding to other phospholipids such as PI(4)P, PA and PE on ELISA. The reactivities of the MAbs with water soluble haptens such as inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were examined by inhibition of liposome lysis. Among three clones examined, only AM-212 showed considerable reactivity with Ins(1,4,5)P3 while also showing weak cross-eactivity with fructose 1,6-diphosphate and inositol 1,4-bisphosphate. No cross-reaction with other structural analogs such as inositol 1-monophosphate and inositol 2-monophosphate was observed. © 1988.