Cytoskeleton in vitro: preparation of isolated cytoskeletons with three-dimensional architecture.
Cytoskeletons with three-dimensional architecture were isolated from cultured normal rat kidney cells. The preparation procedure consisted of Triton-demembranization of suspended cells followed by differential and sucrose density gradient centrifugation. By using higher (0.5%) and lower (0.1%) Triton concentrations for demembranization, two kinds of isolated cytoskeletons (CSK), called H-CSK and L-CSK, respectively, were prepared. H-CSK and L-CSK displayed unique morphology and protein composition. Three classes of cytoskeletal filaments, microfilaments, intermediate filaments, and microtubules were shown to be major components in the electron microscopic images of the H-CSK. Stereoscopic electron microscopy of the H-CSK, dried by the critical point method, revealed that the cytoskeletal filaments are arranged in three-dimensional configurations even after isolation in vitro. Two-dimensional gel electrophoresis demonstrated that the H-CSK was composed mainly of actin, tubulin, and vimentin, reflecting its basic architecture. Electron microscopic images of L-CSK were more intricate than images of the H-CSK and showed, in addition to the filament types discussed above, anastomosing networks of short filamentous structures. These short filaments, with diameters of 3-8 nm and lengths of 30-150 nm, seemed to cross-link other elements of the cytoskeleton. The morphology of these short filaments resembles that of microtrabeculae observed in situ. Two-dimensional gels of the L-CSK showed over 100 protein spots when the gels were stained by the silver method. Subsequent treatment of the L-CSK with 0.5% Triton removed the microtrabeculae-like materials leaving as a residue the basic cytoskeleton similar to the H-CSK. Our observations indicate that microtrabeculae are composed of heterogenous proteins associated, in some instances, with a core structure of actin.
Kuroda
M
M
Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki.
Porter
K R
KR
D000818:Animals; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000302:isolation & purification; D003599:Cytoskeleton / Q000032:analysis* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000648:ultrastructure; D007668:Kidney / Q000648:ultrastructure; D008854:Microscopy, Electron; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats
D003598:Cytoskeletal Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol