Fluorescence-Mediated Visualization of Actin and Myosin Filaments in the Contractile Membrane-Cytoskeleton Complex of Dictyostelium discoideum

Cell Structure and Function

Toshiko Kitanishi-Yumura¹  Shigehiko Yumura¹ 

[1] Biological Institute, Faculty of Science, Yamaguchi University

关键词: membrane-cytoskeleton complex;    actin filament;    myosin filament;    fluorescence staining;    Dictyostelium discoideum;   

DOI  :  10.1247/csf.15.355

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(42)Cited-By(13)An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of micro filament-stabilizing solution squirted from a syringe. The cell membranesleft on the coverslip were immediately stained with tetramethylrhodamine- conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membraneand cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dotlike structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.

【 授权许可】

Unknown