Isolation of cytoskeletons from synchronized plant cells: the interphase microtubule array utilizes multiple tubulin isotypes

摘要: Biochemical analysis of the plant cytoskeleton has been hampered by an inability to isolate anything more than cytoskeletal fragments. Methods for isolating entire detergent-resistant cytoskeletons from carrot suspension cells are now reported. This enables expression tubulin isotypes be studied in functional microtubular arrays, freed soluble pool detergent extraction. When osmotically cushioned with 0.4 M-sorbitol, microtubule-stabilizing buffer and dimethyl-sulphoxide, protoplasts can extracted mild detergent, without fragmenting. Cytoskeletons isolated sucrose density centrifugation shown electron fluorescence microscopy contain a complex meshwork three major fibrous sytems: F-actin, microtubules bundles 7 nm fibrils. Plant assemble into one four microtubule arrays depending upon phase cell cycle. Previous work had established that interphase contained multiple isotypes. However, tubulins taxol assembly vitro , which need not reflect patterns usage array vivo . To address this problem, blocked were converted their determined immunoblotting two-dimensional gels. confirmed all alpha beta identified on gels utilized array. Note: Address reprints