Yuri Nishino

Curved EFC/F-BAR-Domain Dimers Are Joined End to End into a Filament for Membrane Invagination in Endocytosis

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 approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A 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.

Antibodies against Muscle-Specific Kinase Impair Both Presynaptic and Postsynaptic Functions in a Murine Model of Myasthenia Gravis

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

Exon 3 splicing and mutagenesis identify residues influencing cell surface density of heterologously expressed silkworm (Bombyx mori) glutamate-gated chloride channels.

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for l-glutamate and ivermectin (IVM); however, Imax (the maximum l-glutamate– and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest l-glutamate– and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on l-glutamate and IVM actions. Binding assays using [3H]IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced Bmax but not Kd values of [3H]IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the l-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2–TM3 linker, influencing the receptor response.

Reconstitution of the human chaperonin CCT by co-expression of the eight distinct subunits in mammalian cells

The eukaryotic cytosolic chaperonin CCT (chaperonin-containing TCP-1) assists folding of newly synthesized polypeptides. The fully functional CCT is built from two identical rings, each composed of single copies of eight distinct subunits. To study the structure and function of the CCT complex and the role of each subunit, a rapid and efficient method for preparing a recombinant CCT complex is needed. In this work, we established an efficient expression and purification method to obtain human recombinant CCT. BHK-21 cells were infected with a vaccinia virus expressing T7 RNA polymerase and transfected with eight plasmids, each encoding any one of the eight CCT subunits in the T7 RNA polymerase promoter/terminator unit. The CCT1 subunit was engineered to carry a hexa-histidine tag or FLAG tag in the internal loop region. Three days later, cells were harvested for purification of the CCT complex through tag-dependent affinity chromatography and gel filtration. The purified recombinant CCT complexes were indistinguishable from the endogenous CCT purified from HeLa cells in terms of morphology and function. In conclusion, the co-expression system established in this study should be a simple and powerful tool for reconstitution of a large multi-subunit complex.

Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.

Purification and visualization of encephalomyocarditisvirus synthesized by an in vitro protein expression system derived from mammalian cell extract

Virus particles are promising vehicles and templates for vaccination, drug delivery and material sciences. Although infectious picornaviruses can be synthesized from genomic or synthetic RNA by cell-free protein expression systems derived from mammalian cell extract, there has been no direct evidence that authentic viral particles are indeed synthesized in the absence of living cells. We purified encephalomyocarditis virus (EMCV) synthesized by a HeLa cell extract-derived, cell-free protein expression system, and visualized the viral particles by transmission electron-microscopy. The in vitro-synthesized EMCV particles were indistinguishable from the in vivo-synthesized particles. Our results validate the use of the cell-free technique for the synthesis of EMCV particles.

Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS)

Casein micelles are present in bovine milk as colloidal particles with diameters of 20-600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution. The results confirmed that the average casein micelle diameter was about 140 nm, and that the CCP diameter in casein micelles was about 2-3 nm, with an average diameter of 2.3 nm. The distribution of CCP in casein micelles was not uniform, with an average interval between CCPs of about 5.4 nm. Areas containing no black particles (attributed to CCP) were present, with an average size of about 19.1 nm. Considering previous reports, these areas possibly correspond to pores or cavities filled with water. Based on differences in the density of structures in casein micelles, we estimated that some of the casein aggregates were able to connect with CCP in a string.

Microstructural observation of fuel cell catalyst inks by Cryo-SEM and Cryo-TEM

In order to improve the electricity generation performance of fuel cell electric vehicles, it is necessary to optimize the microstructure of the catalyst layer of a polymer electrolyte fuel cell. The catalyst layer is formed by a wet coating process using catalyst inks. Therefore, it is very important to observe the microstructure of the catalyst ink. In this study, the morphology of carbon-supported platinum (Pt/C) particles in catalyst inks with a different solvent composition was investigated by cryogenic scanning electron microscopy (cryo-SEM). In addition, the morphology of the ionomer, which presumably influences the formation of agglomerated Pt/C particles in a catalyst ink, was investigated by cryogenic transmission electron microscopy (cryo-TEM). The results of a cryo-SEM observation revealed that the agglomerated Pt/C particles tended to become coarser with a higher 1-propanol (NPA) weight fraction. The results of a cryo-TEM observation indicated that the actual ionomer dispersion in a catalyst ink formed a network structure different from that of the ionomer in the solvent.

Development of a cryo-SEM system enabling direct observation of the cross sections of an emulsion adhesive in a moist state during the drying process.

In order to analyse the internal structures of multi-component fluid materials such as emulsions (including the inter-particle spacing) by cryo-electron microscopy, it is necessary to observe their smooth cross-sectional surfaces over wide areas. We have developed a system that involves the following steps: preservation of the structure of an emulsion adhesive using freeze fixation in its normal (moist) state and during the drying process after being coated, preparation of cross sections of the internal structure using a cryo-ultramicrotome and then transferral of the cross sections into a cryo-scanning electron microscope for observation via a cryo-transfer system. This system allows the direct observation of the cross sections of emulsions and of several fluid materials.

Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.