Sawaguchi A, McDonald K L, Karvar S, Forte J G
Department of Molecular and Cell Biology, University of California, 241 LSA, Berkeley, CA 94720-3200, USA.
J Microsc. 2002 Dec;208(Pt 3):158-66. doi: 10.1046/j.1365-2818.2002.01085.x.
A newly designated procedure for high-pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel-coated aluminium plates for conventional subsequential cryoimmobilization by high-pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron-dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine-treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H+/K+-ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H+/K+-ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron-dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high-pressure freezing of primary culture cells.
一种新指定的原代培养细胞高压冷冻程序能提供兔胃壁细胞出色的超微结构。将分离的壁细胞培养在基质胶包被的铝板上，以便通过高压冷冻进行常规后续冷冻固定。与传统化学固定相比，不同细胞器（高尔基体、线粒体、多囊泡体等）的超微结构得到了很好的保存。具体而言，微绒毛和顶端下细胞质内清晰可见肌动蛋白丝。细胞骨架系统的另一个显著发现是小管泡之间大量存在微管。有趣的是，一些微管似乎与小管泡相关联。在西咪替丁处理的静息壁细胞顶端膜空泡周围观察到大量电子致密的被膜小窝和小泡，这与静息状态下活跃的膜摄取一致。在小管泡膜上可见H⁺/K⁺-ATP酶的免疫金标记。用组胺刺激时，培养的壁细胞会发生形态转变，导致顶端膜空泡大幅扩张。H⁺/K⁺-ATP酶的免疫金标记不仅出现在扩张的顶端质膜空泡的微绒毛上，也出现在电子致密的被膜小窝中。目前的研究结果为培养的胃壁细胞中的囊泡膜运输提供了线索，并确保了原代培养细胞高压冷冻新程序的实用性。
