CYTOSKELETAL F-ACTIN PATTERNS QUANTITATED WITH FLUORESCEIN ISOTHIOCYANATE-PHALLOIDIN IN NORMAL AND TRANSFORMED-CELLS
CYTOSKELETAL F-ACTIN PATTERNS QUANTITATED WITH FLUORESCEIN ISOTHIOCYANATE-PHALLOIDIN IN NORMAL AND TRANSFORMED-CELLS
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DOI：
10.1073/pnas.77.11.6624
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
POLLACK, R
中科院分区：
文献类型：
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作者：
VERDERAME, M;ALCORTA, D;POLLACK, R
肌动蛋白在培养的成纤维细胞组织成一个复杂的纤维。用肌动蛋白抗体观察到的组织模式与用异硫氰酸荧光素-鬼笔环肽（FL-鬼笔环肽）观察到的组织模式相似但不相同，所述荧光素-鬼笔环肽是一种以2.7 × 10 - 6的解离常数与F-肌动蛋白聚合物结合的化学物质。10-7 M. F1-鬼笔环肽揭示了转化细胞具有比正常细胞更少、更细和更短的含F-肌动蛋白的结构。单细胞的双色荧光显微镜显示，F-phalloidin的F-肌动蛋白染色挑选出的细胞骨架电缆比抗体肌动蛋白更尖锐，由于减少细胞内的背景荧光。这种改进的分辨率允许将细胞FL-鬼笔环肽模式分选为4类，其组织范围从90%的细胞质被大电缆占据到没有可检测电缆。对正常和转化细胞系之间模式分布的可再现差异进行定量。FL-鬼笔环肽，连同基于罗丹明的SV 40肿瘤抗原的间接抗体，揭示了在中间转化的3 T3 [小鼠]细胞系中模式改变的程度和SV 40核抗原表达之间的直接关系。
Actin in cultured fibroblasts is organized into a complex set of fibers. Patterns of organization visualized with antibody to actin are similar but not identical to those visualized with fluorescein isothiocyanate-phalloidin (Fl-phalloidin), a chemical that binds to F-actin polymer with a dissociation constant of 2.7 .times. 10-7 M. Fl-phalloidin reveals that transformed cells have fewer, finer and shorter F-actin-containing structures than do normal cells. Two-color fluorescence microscopy of single cells reveals that F-actin staining by Fl-phalloidin picks out the cytoskeletal cables more sharply than does antibody to actin, due to a reduced intracellular background fluorescence. This improved resolution permits sorting of cellular Fl-phalloidin patterns into 4 classes ranging in organization from 90% of the cytoplasm occupied by large cables to the absence of detectable cables. Reproducible differences in pattern distributions between normal and transformed cell lines were quantitated. Fl-phalloidin, together with rhodamine-based indirect antibody to SV40 tumor antigen, reveals a direct relationship between the degree of pattern change and SV40 nuclear antigen expression in intermediate transformed 3T3 [mouse] cell lines.