Celica Biomedical Center, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
Endocrinology. 2013 Mar;154(3):1235-46. doi: 10.1210/en.2012-2022. Epub 2013 Jan 31.
In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.
在这项研究中，我们使用活细胞免疫细胞化学和共焦显微镜研究了在称为膜草坪的简化系统中单囊泡的释放。草坪是通过使分化的垂体催乳素 (PRL) 分泌细胞暴露于低渗切变力来制备的。免疫标记的三元可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 复合物的密度大约是结合复合蛋白的 PRL 阳性、草坪驻留囊泡的 10 倍；这表明并非所有囊泡都与三元 SNARE 复合物相关。然而，草坪驻留的 PRL 囊泡与特定的 SNARE 蛋白相对较好地共定位：突触融合蛋白 2（35%）、突触素 1（22%）和 25kDa 突触体相关蛋白（6%）。为了研究囊泡排放，我们制备了草坪驻留的囊泡，这些囊泡来源于心房利钠肽标记有祖母绿荧光蛋白（ANP.emd）的转染细胞，这些囊泡可标记囊泡。由于大约 40%的 ANP.emd 加载囊泡被细胞外 PRL 抗体标记，因此这些囊泡保持了与外部的结构通道。通过 ANP.emd 荧光强度的下降监测草坪驻留囊泡的货物释放，与完整细胞中的释放相似。SNARE 蛋白可能是这些囊泡钙依赖性释放所必需的。这是因为表达显性负 SNARE 肽，该肽干扰 SNARE 复合物的形成，使细胞内每个 PRL 阳性斑点的数量（置于细胞外的 PRL 抗体）显著减少，从 58 ± 9 减少到 4 ± 2。在显性负 SNARE 处理的细胞中，PRL 阳性区域从 0.259 ± 0.013 减少到 0.123 ± 0.014 μm(2)，这与细胞外 PRL 抗体进入囊泡腔的受阻一致。这些结果表明囊泡释放受 SNARE 介导的融合孔扩大调节。
