ISOLATION AND CHARACTERIZATION OF CYTOSKELETONS FROM COTTON FIBER CYTOPLASTS

Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to techn ical advances in the isolation and purification of anucleate cytoskele tons from animal cells. As a step towards characterizing protein compo nents of the plant cytoskeleton, we have isolated cytoskeletons from c ytoplasts (anucleate protoplasts) prepared from cotton fiber cells gro wn in ovule culture. Cytoplasts isolated into a hypertonic, Ca2+-free medium at pH 6.8 retained internal structures after extraction with th e detergent, Triton X-100. These structures were shown to include micr otubule and microfilament arrays by immunofluorescence and electron mi croscopy. Actin and tubulin were the only abundant proteins in these p reparations, suggesting that microfilaments and microtubules were the major cytoskeletal elements in the isolated cytoskeletons. The absence cf additional, relatively abundant proteins suggests that (a) other c ytoskeletal arrays potentially present in fiber cells (e.g., intermedi ate filaments) were either lost during detergent extraction or were mi nor components of the fiber cell cytoskeleton, and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubu lin were not required to maintain microtubules and microfilaments in o rganized structures.