Mundy Dorothy I, Machleidt Thomas, Ying Yun-shu, Anderson Richard G W, Bloom George S
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA.
J Cell Sci. 2002 Nov 15;115(Pt 22):4327-39. doi: 10.1242/jcs.00117.
Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 microm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call 'cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.
利用活细胞延时显微镜技术研究了稳定表达的CHO细胞中窖蛋白-1-GFP的运输情况。多项细胞学和生化检测证实，窖蛋白-1-GFP是内源性窖蛋白-1的可靠标记物。在稳态下，大多数窖蛋白-1-GFP要么位于与内陷小窝相关的细胞表面，要么位于窖穴小体中的中心体附近。活细胞荧光成像表明，虽然细胞表面小窝中的许多窖蛋白-1-GFP相对静止，但细胞内部存在大量高度移动的窖蛋白-1-GFP阳性囊泡。这些囊泡的移动速度在0.3-2微米/秒之间，当用诺考达唑使微管解聚时，移动停止。在没有微管的情况下，细胞表面内陷的小窝增加了两倍多，并排列成线性阵列。相比之下，用拉春库林A完全解聚肌动蛋白细胞骨架会引发窖蛋白阳性结构向细胞中心体区域的快速大量移动。因此，CHO细胞的小窝膜系统似乎由三个含有窖蛋白-1的区室组成。这些包括被皮质肌动蛋白丝限制在细胞表面的小窝、中心体周围的窖穴小体和窖泡，我们称之为“窖管”，它们沿着微管在细胞表面和窖穴小体之间持续双向移动。窖管的行为表明它们充当小窝和窖穴小体之间的运输中间体。
