Vikstrom K L, Borisy G G, Goldman R D
Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago, IL 60611.
Proc Natl Acad Sci U S A. 1989 Jan;86(2):549-53. doi: 10.1073/pnas.86.2.549.
A procedure was developed for the conjugation of vimentin with biotin. Biotinylated vimentin was then microinjected into BHK-21 cells and the fate of the labeled protein was determined at various times postinjection by indirect immunofluorescence. Microinjected vimentin could be traced through a specific sequence of morphological changes ultimately resulting in the formation of a filamentous network. The injected protein was first detected in spots dispersed throughout the cytoplasm. Subsequently, these spots appeared to cluster near the nucleus where they merged into a diffuse "cap." This cap coincided with a concentration of endogenous intermediate filaments and eventually gave rise to a filamentous network that was coincident with the endogenous intermediate filament network as determined by double-label immunofluorescence. The results indicate that the incorporation of exogenous vimentin into a filamentous network is initiated in a perinuclear region and progresses in a polarized fashion toward the cell surface.
开发了一种将波形蛋白与生物素偶联的方法。然后将生物素化的波形蛋白显微注射到BHK - 21细胞中，并在注射后不同时间通过间接免疫荧光法确定标记蛋白的命运。显微注射的波形蛋白可以通过一系列特定的形态变化被追踪到，最终形成丝状网络。注射的蛋白首先在分散于整个细胞质中的斑点中被检测到。随后，这些斑点似乎聚集在细胞核附近，在那里它们合并成一个弥散的“帽”。这个帽与内源性中间丝的聚集相吻合，最终产生一个丝状网络，通过双标记免疫荧光法确定，该网络与内源性中间丝网络重合。结果表明，外源波形蛋白掺入丝状网络是在核周区域开始的，并以极化方式向细胞表面发展。
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