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

Cytoplasts from cotton (Gossypium hirsutum L.) fiber cells retain microtubu le and microfilament cytoskeletons through extraction with non-ionic deterg ent and ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N' -tetraacetic acid. Tubulin and actin are the most abundant proteins in extracted cytopla sts; however, many other less abundant proteins are also present. To determ ine if minor proteins were associated with the cytoskeleton, microtubules a nd microfilaments were selectively removed from extracted cytoplasts by det ergent extraction in an alkaline Ca2+ solution. Under these extraction cond itions, microtubules and microfilaments were fragmented and depolymerized u nless previously stabilized by taxol and phalloidin. Associated proteins we re identified by their loss in conjunction with either microtubules or micr ofilaments. As judged by sodium dodecyl sulfate-polyacrylamide gel electrop horesis, one protein, of roughly 115 kDa, appeared to be associated with mi crofilaments since it was present in Ca2+-extracted preparations only when microfilaments were stabilized with phalloidin. The failure of most minor p roteins to associate with microtubules and microfilaments suggests that cau tion must be used when interpreting co-isolation as evidence for an associa tion of low abundance proteins with cytoskeletons. USDA-ARS (C) 2000 Publis hed by Editions scientifiques et medicales Elsevier SAS.