Abstract
The cytoskeleton is fundamental to many cellular functions including cell proliferation, differentiation, adhesion, and migration. It is composed of actin, microtubules, intermediate filaments, and integrin cell surface receptors, which form focal adhesions with the extracellular matrix. These elements are highly integrated in the cell providing a rigid network of interconnected cables and protein scaffolds, which generate force and mechanical support to maintain cell shape and movement. However, the cytoskeleton is not just a simple compilation of static filaments that dictate cell adhesion and morphology-it is highly plastic with the inherent ability to assemble and disassemble in response to diverse and complex cellular cues. Thus, biochemical and proteomic methods are needed to better understand the cytoskeleton network and its dynamic signal transduction functions in health and disease. This chapter describes methods for the biochemical enrichment and mass spectrometry-based proteomic analyses of the cytoskeletome. We also detail how these methods can be used to investigate the cytoskeletome of migrating cells and their purified pseudopodia membrane projections.