Co-sedimentation of Actin, Tubulin andMembranes in the Cytoskeleton Fractions from Peas and Mouse 3T3 Cells

Abstract

Pea stem tissue (Pisum sativum L. var. Alaska) was homogenized in a recently-developed cytoskeleton-stabilizing buffer, CSB,(Abe and Da vies, 1991) and homogenates electrophoresed and blotted on to membranes. Blots probed individually withantibodies to actin, alpha-tubulin, and beta-tubulin, revealed bands with apparent molecular weights of 42, 46, and 48–50 kDa,respectively. Blots probed with all three antibodies simultaneously revealed all three bands which could be distinguished in thesame lane. Homogenates of mouse 3T3 cells yielded an actin band at about 42 kDa, but both alpha- and beta-tubulin appeared atabout 50 kDa and thus could not be distinguished on blots probed simultaneously. This ‘triple-blotting technique’ was, therefore,suitable for pea tissue, but not for mouse tissue. In pea tissue, sedimentable tubulin and actin were found maximally in the 4000 ×g pellet and less in successive 15000 and l00000×g pellets. Both EGTA and Mg2+ which had been found earlier to beessential for stability of the actin cytoskeleton as revealed by fluorescence microscopy, were essential for co-sedimentation of actinand tubulin. In contrast to the results with pea stems, only the actin component of the cytoskeleton could be isolated from mouse 3T3 cells using CSB. Pea tissue was homogenized in CSB without PTE and the resulting cytoskeletal pellets resuspended in actin- or tubulin-solubilizing buffers with and without PTE. In the absence of PTE, the buffers solubilized their appropriate cytoskeletal protein, but little of the other protein, while in the presence of PTE both proteins were quite effectively solubilized by both buffers. Incontrast, in CSB with or without PTE, both proteins remained in the sedimentable fraction. These results, taken together withother evidence, indicate that microtubules, as well as microfilaments are important components of the sedimentable cytoskeletonfraction of peas and that the membrane system is intimately involved in organization of the cytoskeleton in peas.