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Journal of Cell Science, Vol 88, 225-230, Copyright © 1987 by Company of Biologists

Submitted on May 11, 1987
Accepted on June 17, 1987

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

PATRICK J. HUSSEY 1, JAN A. TRAAS 2, KEITH GULL 3, and CLIVE W. LLOYD 2

1 Department of Cell Biology, The John Innes Institute, Colney Lane, Norwich NR4 7UH, UK; The Biological Laboratory, The University of Kent at Canterbury, Canterbury, Kent, UK; Department of Genetics and Cell Biology, University of Minnesota, St Paul, Minnesota, USA
2 Department of Cell Biology, The John Innes Institute, Colney Lane, Norwich NR4 7UH, UK
3 The Biological Laboratory, The University of Kent at Canterbury, Canterbury, Kent, UK

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 0.4 M-sorbitol, microtubule-stabilizing buffer and dimethyl-sulphoxide, 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.

Note:
Address for reprints

Key words: cortical microtubules, carrot cells, tubulin isotypes, cytoskeletons, synchronization

Submitted on May 11, 1987
Accepted on June 17, 1987




This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Chan, C. G. Jensen, L. C. W. Jensen, M. Bush, and C. W. Lloyd
The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules
PNAS, December 21, 1999; 96(26): 14931 - 14936.
[Abstract] [Full Text] [PDF]


© The Company of Biologists Ltd 1987