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

Abstract

ABSTRACT 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 the expression of tubulin isotypes to be studied in functional microtubular arrays, freed of the soluble pool by detergent extraction. When osmotically cushioned with 0·4M-sorbitol, microtubule-stabilizing buffer and dimethylsulphoxide, carrot protoplasts can be extracted by mild detergent, without fragmenting. Cytoskeletons isolated by sucrose density centrifugation are shown by electron and fluorescence microscopy to contain a complex meshwork of three major fibrous sytems: F-actin, microtubules and bundles of 7 nm fibrils. Plant cells can assemble tubulin into one of four microtubule arrays depending upon the phase of the cell cycle. Previous work had established that interphase cells contained multiple tubulin isotypes. However, the tubulins had been isolated by taxol assembly in vitro, which need not reflect patterns of usage by the interphase microtubule array in vivo. To address this problem, cells blocked in interphase were converted to cytoskeletons and their usage of tubulin isotypes determined by immunoblotting two-dimensional gels. This confirmed that all of the alpha and beta isotypes that can be identified on two-dimensional gels of carrot suspension cells are utilized by the interphase microtubule array.