Nakata T, Hirokawa N
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
J Neurosci. 1992 Jun;12(6):2186-97. doi: 10.1523/JNEUROSCI.12-06-02186.1992.
We have studied the organization of the cytoskeleton in both unstimulated and stimulated cultured chromaffin cells, as well as its relationship with their secretory process by exocytosis. We found the spatial heterogeneity in the intensity of cortical rhodamine-phalloidin staining within a cell. The overall staining pattern or intensity was minimally altered after stimulation, although dopamine-beta-hydroxylase (DBH) antigen, a marker for the chromaffin granule membrane, was exposed preferentially on the plasma membrane areas with lower intensity of rhodamine-phalloidin staining. Using the quick-freeze, deep-etch technique, we found the heterogeneity in the organization of cortical cytoskeletal networks--some regions have actin filament bundles running parallel to the plasma membrane interspersed between granules and the plasma membrane, while others have few actin filaments beneath the plasma membrane before stimulation. Actin filaments were rarely observed in the inner cytoplasm. We did not observe the overall change in its organization after stimulation. Double-label immunogold EM using anti-DBH antibody and anti-actin antibody combined with statistical analysis showed that (1) DBH was exposed on the plasma membrane preferentially where actin was sparse after stimulation (significant at less than 0.1%), although (2) regions having sparse actin were not always the sites for DBH exposure, and (3) the cortical actin zone was sometimes disrupted at the DBH-exposed sites after stimulation. The present data suggested that (1) secretion is related to heterogeneous organization of cortical cytoskeleton after stimulation and (2) massive synchronized reorganization of the cytoskeleton in the whole cell is not necessary for secretion, although small changes of the cytoskeleton might occur under local regulation at each exocytotic site at the moment of the release.
我们研究了未受刺激和受刺激的培养嗜铬细胞中细胞骨架的组织情况，以及其与通过胞吐作用的分泌过程之间的关系。我们发现细胞内皮质罗丹明 - 鬼笔环肽染色强度存在空间异质性。刺激后，整体染色模式或强度变化极小，尽管嗜铬颗粒膜标记物多巴胺 -β- 羟化酶（DBH）抗原优先暴露于罗丹明 - 鬼笔环肽染色强度较低的质膜区域。使用快速冷冻、深度蚀刻技术，我们发现皮质细胞骨架网络的组织存在异质性——一些区域有与质膜平行排列的肌动蛋白丝束散布在颗粒和质膜之间，而另一些区域在刺激前质膜下方几乎没有肌动蛋白丝。在内质中很少观察到肌动蛋白丝。刺激后我们未观察到其组织的整体变化。使用抗DBH抗体和抗肌动蛋白抗体结合统计分析的双标记免疫金电子显微镜显示：（1）刺激后DBH优先暴露于肌动蛋白稀疏的质膜上（在小于0.1%时具有显著性），尽管（2）肌动蛋白稀疏的区域并不总是DBH暴露的位点，并且（3）刺激后皮质肌动蛋白区有时在DBH暴露位点处被破坏。目前的数据表明：（1）分泌与刺激后皮质细胞骨架的异质组织有关，并且（2）尽管在释放瞬间每个胞吐位点在局部调节下细胞骨架可能会发生小的变化，但整个细胞中细胞骨架的大规模同步重组对于分泌并非必要。
