Selective extraction of cotton fiber cytoplasts to identify cytoskeletal-associated proteins

• Published in: Plant physiology and biochemistry Vol. 38; no. 3; pp. 193 - 199
• Main Authors: Andersland, John M, Triplett, Barbara A
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01.03.2000; Elsevier
• Subjects: ACTIN; ACTINA; ACTINE; Biological and medical sciences; Cell biochemistry; Cell physiology; CELL WALLS; CELLS; CELLULE; CELULAS; cytoskeleton; FIBRE CROPS; Fundamental and applied biological sciences. Psychology; GOSSYPIUM HIRSUTUM; microfilaments; microtubules; PARED CELULAR; PAROI CELLULAIRE; Plant physiology and development; PLANTAS TEXTILES; PLANTE A FIBRES; PROTEINAS; PROTEINE; PROTEINS; tubulin; cytoskeleton; DIC, differential interference contrast; tubulin; IF, intermediate filaments; microtubules; TRIS, 2-amino-2-(hydroxymethyl)-1,3-propanediol; MTs, microtubules; MFs, microfilaments; PMSF, phenylmethylsulfonyl fluoride; Actin; DAPI, 4,6-diamidino-2-phenylindole; microfilaments; PIPES, piperazine-N,N'-bis[2-ethanesulfonic acid]; EGTA, ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid; Malvaceae; Cytoplast; Plant fiber; Microfilament; Method; Fiber crop; Protein; Dicotyledones; Angiospermae; Affinity; Cytoskeleton; Isolation; Spermatophyta; Gossypium hirsutum; Microtubule
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/S0981-9428(00)00734-8

Cytoplasts from cotton ( Gossypium hirsutum L.) fiber cells retain microtubule and microfilament cytoskeletons through extraction with non-ionic detergent and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid. Tubulin and actin are the most abundant proteins in extracted cytoplasts; however, many other less abundant proteins are also present. To determine if minor proteins were associated with the cytoskeleton, microtubules and microfilaments were selectively removed from extracted cytoplasts by detergent extraction in an alkaline Ca 2+ solution. Under these extraction conditions, microtubules and microfilaments were fragmented and depolymerized unless previously stabilized by taxol and phalloidin. Associated proteins were identified by their loss in conjunction with either microtubules or microfilaments. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one protein, of roughly 115 kDa, appeared to be associated with microfilaments since it was present in Ca 2+-extracted preparations only when microfilaments were stabilized with phalloidin. The failure of most minor proteins to associate with microtubules and microfilaments suggests that caution must be used when interpreting co-isolation as evidence for an association of low abundance proteins with cytoskeletons.