Methods for isolation and analysis of the cytoskeleton

This chapter discusses methods for the isolation and analysis of the cytoskeleton (CSK). The plant CSK consists of microfilaments (MFs) and microtubules (MTs) interchanging dynamically with their corresponding monomeric proteins, actin and tubulin, and perhaps of intermediate filaments. Monomeric actin and tubulin have been isolated from many plant tissues and described. The chapter focuses on the isolation of the CSK itself and deals with solubilized monomers. The first methods for isolating relatively intact CSK in amounts sufficient for biochemical analysis employed protoplasts from carrot suspension cells (Hussey et al). These pioneering methods suffer from two potential drawbacks: (1) they are suitable only for tissues that yield protoplasts easily and (2) the conditions needed to release protoplasts can themselves cause substantial changes in the CSK and thus lead to artifacts (Tan and Boss). Relatively little is known about actin-binding proteins (ABPs) and tubulin-binding proteins (TBPs) and even less about MF-associated proteins (MFAPs) and MT-associated proteins (MTAPs) because the isolation of actin as MFs and tubulin as MTs is a prerequisite for identifying MF-binding proteins (MFBPs), MTAPs, and any other cellular components that might associate in vivo with the CSK. © 1995, Academic Press, Inc. All rights reserved