Etsuko Suzaki

Human Coronavirus 229E Binds to CD13 in Rafts and Enters the Cell through Caveolae

ABSTRACT CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl β-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

Video-rate dynamics of exocytotic events associated with phagocytosis in neutrophils.

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunners gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.

Characterization of proteins secreted from a Type III secretion system of Edwardsiella tarda and their roles in macrophage infection

The Type III secretion system is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. We identified the secreted proteins of the Type III secretion system by comparing the wild-type strain and the Type III mutant mET1229. The wild-type strain secreted 55, 25, and 22 kDa proteins into the culture supernatant, whereas the Type III mutant did not. These proteins were identified as EseB, EseC, and EseD and are similar in sequence to Salmonella SseB, SseC, and SseD that function as a translocon. The EseB, EseC, and EseD knockout mutants did not replicate in murine macrophages, suggesting that these proteins are essential for intracellular replication of E. tarda. Highest secretion of EseBCD proteins was observed when bacterial cells were cultured in neutral and alkaline pHs but not in acidic pH. When the pH of the phagosomes was examined using an acidotropic probe, the phagosomes containing the wild-type strain showed neutral pH, whereas those containing the Type III mutant exhibited acidic pH. These results suggest that the Type III-dependent interference with formation of the acidic environment in phagosomes is essential for intracellular replication of bacteria in murine macrophages.

Quantitative analysis of exocytosis visualized by a video-enhanced light/fluorescence microscope reveals two distinct components of exocytosis in RBL-2H3 cells

Rat basophilic leukemia (RBL‐2H3) cells, which exhibit Ca2+‐dependent secretion of granules when stimulated with antigen or the Ca2+‐ionophore A23187, were observed under a video‐enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31–8425, Ro31–8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL2H3 cells.

Quantitative analysis of superoxide anion generation in living cells by using chemiluminescence video microscopy

Superoxide anions (O2-) generated by rabbit neutrophils were detected and quantified by a video microscope equipped with a photon-counting camera. One count obtained by this system was equivalent to 59 amol of O2-. Maximum O2- production was observed at 6-8 min after stimulation and was estimated as 1.9 fmol/min per cell on the average.

The Golgi apparatus of goblet cells in the mouse descending colon: three-dimensional visualization using a confocal laser scanning microscope

Abstract. The three-dimensional structure of the Golgi apparatus was studied in goblet cells in lectin-stained sections of the mouse descending colon by using a confocal laser scanning microscope. In the lower part of the crypt, the Golgi apparatus formed a dome- or globe-like structure in the supranuclear region. The wall of the dome had some holes, one of which usually faced toward the nucleus and others toward the apical cytoplasm. Mucous granules seemed to be initially released into the interior of the dome and transported toward the apical cytoplasm through the holes. In the upper part of the crypt, on the other hand, the Golgi apparatus formed a cup- or funnel-like structure with a larger opening toward the cell apex and a smaller opening toward the nucleus. A large mass of mucous granules occupied the inside of the cup to the apical cytoplasm. It is thought that the accumulation of mucous granules enlarges holes at the ceiling of the dome to form a large opening, which makes the configuration of the Golgi apparatus cup-shaped.

Visualization and dynamic size evaluation of nanoparticles in solution by single optical fiber-illuminated video microscope analysis

AIM It is hoped that nanoparticles will become ever more useful in the development of nanomedicine. To evaluate the behavior of nanoparticles in solution, we aimed to establish a single optical fiber-illumination method that is easy to integrate with a conventional microscope at low cost. METHODS Solutions of gold nanoparticles and carbon nanotubes were analyzed in a single optical fiber-illuminated video microscope and the tracks of Brownian motion of these nanoparticles were traced using video images. Their diffusion coefficient was measured by the mean square displacement of the movement. Using the diffusion coefficient in the Stokes-Einstein equation, the hydrodynamic diameter of the nanoparticles in solution was evaluated. RESULTS The visualization of gold nanoparticles clearly in a high signal-to-noise ratio was achieved. The evaluated particle sizes of gold nanoparticles were similar to those obtained by a transmission electron microscope and the aggregation process of the carbon nanotubes following incubation was also observed and similar size estimation of the aggregates was performed. CONCLUSION The single fiber-illumination method was applicable to visualize nanoparticle movement clearly and to estimate their sizes in solution. This simple method is suitable for the in situ observation of the nanoparticle-binding process to target cells.