Osamu Katsumata

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

J. Neurochem. (2011) 116, 1122–1137.

Differential expression of SNAP-25 family proteins in the mouse brain.

Soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP)‐25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP‐25 family proteins: SNAP‐25a, SNAP‐25b, and SNAP‐23, are expressed in the brain, but little is known about their distributions and functions. In the present study, we generated specific antibodies to distinguish these three homologous proteins. Immunoblot and immunohistochemical analyses revealed that SNAP‐25b was distributed in synapse‐enriched regions throughout almost the entire brain, whereas SNAP‐25a and SNAP‐23 were expressed in relatively specific brain regions with partially complementary expression patterns. SNAP‐25a and SNAP‐25b, but not SNAP‐23, were also present in the axoplasm of nerve fibers. The intracellular localization was also different, and although SNAP‐25b and SNAP‐23 were found primarily in membrane and lipid raft‐enriched fractions of mouse brain homogenates, a substantial amount of SNAP‐25a was recovered in soluble fractions. In PC12 cells, SNAP‐25b was localized to the plasma membrane, but SNAP‐25a and SNAP‐23 were distributed throughout the cytoplasm. The expression and distribution of these three proteins were also differentially regulated in the early postnatal period. These results indicate that the three SNAP‐25 family proteins display a differential distribution in the brain as well as in neuronal cells, and possibly play distinct roles. J. Comp. Neurol. 519:916–932, 2011.

The postsynaptic density protein, IQ-ArfGEF/BRAG1, can interact with IRSp53 through its proline-rich sequence

IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQ-ArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95.

IQ‐ArfGEF/BRAG1 is associated with synaptic ribbons in the mouse retina

IQ‐ArfGEF/BRAG1 is a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), which are implicated in membrane trafficking and actin cytoskeleton dynamics. In this study, we examined the immunohistochemical localization of IQ‐ArfGEF/BRAG1 in the adult mouse retina using light and electron microscopy. IQ‐ArfGEF/BRAG1 was distributed in a punctate manner and colocalized well with RIBEYE in both the outer and inner plexiform layers. Immunoelectron microscopic analysis showed that IQ‐ArfGEF/BRAG1 was localized at the synaptic ribbons of photoreceptors. When heterologously expressed in HeLa cells, IQ‐ArfGEF/BRAG1 was recruited to RIBEYE‐containing clusters and formed an immunoprecipitable complex with RIBEYE. Furthermore, immunoprecipitation analysis showed that anti‐IQ‐ArfGEF/BRAG1 antibody efficiently pulled down RIBEYE from retinal lysates. These findings indicate that IQ‐ArfGEF/BRAG1 is a novel component of retinal synaptic ribbons and forms a protein complex with RIBEYE.

Collagen II containing a Cys substitution for Arg-α1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.

Establishment of a standardized post-embedding method for immunoelectron microscopy by applying heat-induced antigen retrieval.

We have developed a new standardized method for the post-embedding immunoelectron microscopy using the same fixation, antigen retrieval and image contrasting procedures. Tissues were fixed with 4% formaldehyde containing 2.5 mM CaCl(2), 1.25 mM MgCl(2) in a 0.1 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) for 2 h and then with the same fixative composition in 0.1 M HEPES buffer (pH 8.5) overnight at room temperature. Vehicle osmolarity of fixatives was adjusted to 300-330 mOsm by adding glucose. The specimens were dehydrated with dimethylformamide on ice and embedded in LR-White resin. Ultrathin sections were heated in a 20 mM Tris-HCl buffer (pH 9.0) for 1-2 h at 95 degrees C. After immuno-gold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in a 0.1 M phosphate buffer (pH 5.5) for 5 min and with a 1% OsO(4)/0.1 M phosphate buffer (pH 7.4) for 5 min, and then they were double stained with uranyl acetate and lead citrate. The standardized method yielded strong and reproducible immunoreactions for soluble, membrane-bound and filamentous proteins showing an excellent image contrast without destruction of the fine structures.

Intercalated duct cells in the rat parotid gland may behave as tissue stem cells

This study was conducted to determine whether intercalated duct cells in the rat parotid gland have the properties of tissue stem cells. After induction of cellular proliferation by repeated administration of isoproterenol (IPR), a β-adrenergic agonist, proliferation activity in acinar, intralobular, and intercalated ductal cells was quantified using Ki-67 immunohistochemistry. The total number of each type of component cell in a gland was also estimated in the course of IPR treatment. IPR was found to induce proliferation of acinar and intercalated duct cells, but not intralobular duct cells. The total number of acinar cells in a gland on day 5 of IPR treatment was 1.6 times of that at day 0 (baseline). In contrast, the total numbers of intercalated and intralobular duct cells did not change from baseline, indicating a high possibility that the proliferated intercalated duct cells differentiated into acinar cells. On days 2 to 3 of IPR treatment, intercalated duct cells with amylase-positive secretory granules were recognized in a region very close to the acini, and were suspected of being transitional cells from intercalated duct to acinar cells. This quantitative study indicates that intercalated duct cells may have the properties of tissue stem cells upon IPR stimulation.

Electron microscopic localization of 5′-nucleotidase in rat salivary glands

SummaryThe localization of 5′-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5′-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5′-nucleotidase activity and compare it with the traditional lead-ion method.

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure.

Pilt physically interacts with Arf6(Q67L) by pull down (View interaction)

Predominant localization of EFA6A, a guanine nucleotide exchange factor for ARF6, at the perisynaptic photoreceptor processes

EFA6A is a guanine nucleotide exchange factor that is highly expressed in the nervous system with the ability to activate ADP ribosylation factor 6 (ARF6). In this study, we demonstrated the immunohistochemical localization of EFA6A in the adult mouse retina. Strong immunoreactivity for EFA6A was detected predominantly in the outer plexiform layer (OPL), where EFA6A was partially overlapped with dystrophin and synaptophysin. Immunoelectron microscopic analysis revealed that EFA6A was localized predominantly at the perisynaptic processes of photoreceptor terminals without association with synaptic ribbons. These findings suggest that EFA6A-ARF6 pathway may play a specific role at a subcompartment of perisynaptic photoreceptor processes.