Journal Article

Isolation of Cortical MTs from Tobacco BY-2 Cells

Seiji Sonobe, Shushi Yamamoto, Muneki Motomura and Teruo Shimmen

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 2, pages 162-169
Published in print February 2001 | ISSN: 0032-0781
Published online February 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce017
Isolation of Cortical MTs from Tobacco BY-2 Cells

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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We isolated the cortical microtubules (CMTs) from tobacco BY-2 cells to identify their components. By centrifugation of protoplasts homogenized in the presence of taxol, a MT-stabilizing reagent, in a density gradient of Percoll, we obtained membranous vesicles to which MTs forming a sheet-like bundle were attached. Rhodamine-conjugated Ricinus communis agglutinin I (RCA-I), a lectin that bound to the surface of protoplasts, stained these vesicles, indicating that they were plasma membrane (PM) vesicles that retained CMTs. CMTs were released by solubilization of PM vesicles with Triton X-100. A sheet-like array of CMTs was retained even after solubilization of PM vesicles. Immunoblot analysis of the isolated CMTs demonstrated the presence of tubulin, actin, the 65 kDa microtubule-associated protein (MAP) and a 130 kDa RCA-I binding protein. Purification of the isolated CMTs by the temperature dependent disassembly-reassembly cycling method revealed four polypeptides, 190, 120, 85 and 65 kDa, co-assembling with CMTs.

Keywords: Key words: Cortical microtubules — Cross-bridges — Microtubule-associated protein — Plasma Membrane — Tobacco BY-2.; Abbreviations: CMT, cortical microtubules; DTT, dithiothreitol; EGTA, ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid; MAPs, microtubule-associated proteins; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; PM, plasma membrane.

Journal Article.  4816 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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