We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cellfree system involving endothelial PM sheets and isolated biotinlabeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis. The endothelial cells (ECs)3 lining the blood vessel walls are differentiated to mediate the rapid exchange of substances between the plasma and interstitial fluid. The process involving the fission of caveolae from the apical plasma membrane (PM) and fusion with the basolateral PM is termed transcytosis. Caveolae "pinch off " from the apical PM through a process requiring the recruitment of the GTPase dynamin to the caveolar necks as regulated by another protein intersectin (1, 2). Upon fission, caveolae form vesicular carriers that shuttle through the cytosol and deliver their cargo to the subendothelium by fusion with the basolateral PM (3). The basis of caveolar fusion may involve the same machinery as other vesicular carriers, i.e. the SNARE proteins of the syntaxin, synaptobrevin/cellubrevin, and SNAP-23/25 families (4 -7). However, the organization of SNARE proteins and their function in the fusion of caveolae in ECs are incompletely understood. It is known that syntaxin, cellubrevin, SNAP-23, and the cytosolic factors N-ethylmaleimide-sensitive factor (NSF) and ␣-and ␥-SNAP are key components of the endothelial multimolecular transcytotic complex, the assembly of which depends on the membrane fusion ATPase, NSF, which can be inhibited by alkylation of NSF by N-ethylmaleimide (7). Studies have also shown that N-ethylmaleimide interferes with transcytosis in ECs (4, 8). Based on the SNARE hypothesis, membrane fusion occurs when SNARE proteins on opposing membranes form four helix bundles, bringing the membranes in close apposition, thus providing the driving force necessary for fusion (9, 10). Ultrastructural studies have shown several states of association between caveolae and their target PM, varying in proximity, stability, and readiness for fusion (11). However, the pre-fusion states of caveolae have not been experimentally clarified, and the sequence of events responsible for caveolar fusion...

Polymorphonuclear leukocytes (PMNs) can be divided into Gr-1high and Gr-1low subpopulations, but the differences in the functions of these cells in the host are unknown. This study investigated the roles of these two cell populations in the clearance of an intracellular pathogen (Haemophilus influenzae) causing murine peritonitis and pneumonia. Microarray analysis and quantitative real-time PCR analysis of proteose peptone-elicited peritoneal murine PMNs showed that IL-15 mRNA levels were significantly higher in Gr-1high PMNs than in Gr-1low PMNs. In addition, IL-15 was produced only by Gr-1-positive PMNs, especially Gr-1high PMNs. IL-15 was required for efficient clearance of experimental murine H. influenzae pneumonia, as 4 days postinfection lungs from IL-15 knockout mice contained 50- to 100-fold more bacteria than did wild-type mouse lungs. Gr-1 PMN-depleted C57BL/6 mice were more susceptible to H. influenzae pneumonia than were Gr-1 PMN replete C57BL/6 mice or C57BL/6 nude mice, demonstrating that Gr-1 PMNs are important in the clearance of intracellular bacteria. IL-15-activated NK cells killed H. influenzae in PMNs. Flow cytometry confirmed the expression of CD69 on the cell membrane of IL-15-activated NK cells. Our results show that Gr-1high PMNs produce more IL-15 than Gr-1low PMNs, and that IL-15-activated NK cells protect against early infection by H. influenzae.

NF 3 was applied in the reactive ion etching of SiC. The effects of rf power and NF3 pressure on the etching rate and the surface morphology were investigated by means of scanning electron microscopy and atomic force microscopy. A procedure for getting the smooth and residue-free etched surface of SiC with a high etching rate of 87 nm/min was obtained under the conditions such as rf power of 100 W and NF3 pressure ranging from 0.5 to 1 Pa. A rough surface with spikes was obtained under the NF3 pressures higher than 3 Pa. It was found that the repetitive alternating treatment for the spike-formed and rough surface with the down flow etching using NF3 and Ar plasma sputtering enables us to obtain the smooth surface within the scale of ∼300 nm.

Articles you may be interested inHigh-stroke silicon-on-insulator MEMS nanopositioner: Control design for non-raster scan atomic force microscopy Rev. Sci. Instrum. 86, 023705 (2015); 10.1063/1.4907908High-aspect-ratio grooves fabricated in silicon by a single pass of femtosecond laser pulses J. Appl. Phys. 111, 093102 (2012); 10.1063/1.4709726 Fabrication of high-aspect-ratio lightpipesA microprobe having an ultrasharp and high-aspect-ratio stylus was made using a directly bonded silicon-on-insulator ͑SOI͒ wafer. The stylus and cantilever were made of 8 m-thick ͑100͒-Si film and 2 m-thick dioxide film of the SOI wafer with sufficient reproducibility. The cantilever is 100 m in length, 20 m in width, and 1.6 m in thickness; the stylus is 7 m in height with an aspect ratio exceeding 2. The stylus was formed first by reactive ion etching and sharpened by orientation dependent etching with KOH solution. The apex of the stylus is 10 nm in radius of curvature. The present microprobe was found adequate for atomic force microscopy ͑AFM͒ measurement with spatial resolution better than 10 nm in noncontact-mode topographic imaging. The mode of fabrication, mechanical properties of the microprobe, and results of AFM measurement are discussed.