title: Procedures for the biochemical enrichment and proteomic analysis of the cytoskeletome.
abstract: The cell cytoskeleton is composed of microtubules, intermediate filaments, and actin that provide a rigid support structure important for cell shape. However, it is also a dynamic signaling scaffold that receives and transmits complex mechanosensing stimuli that regulate normal physiological and aberrant pathophysiological processes. Studying cytoskeletal functions in the cytoskeleton's native state is inherently difficult due to its rigid and insoluble nature. This has severely limited detailed proteomic analyses of the complex protein networks that regulate the cytoskeleton. Here, we describe a purification method that enriches for the cytoskeleton and its associated proteins in their native state that is also compatible with current mass spectrometry-based protein detection methods. This method can be used for biochemical, fluorescence, and large-scale proteomic analyses of numerous cell types. Using this approach, 2346 proteins were identified in the cytoskeletal fraction of purified mouse embryonic fibroblasts, of which 635 proteins were either known cytoskeleton proteins or cytoskeleton-interacting proteins. Functional annotation and network analyses using the Ingenuity Knowledge Database of the cytoskeletome revealed important nodes of interconnectivity surrounding well-established regulators of the actin cytoskeleton and focal adhesion complexes. This improved cytoskeleton purification method will aid our understanding of how the cytoskeleton controls normal and diseased cell functions.
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Sunkyu
authors.initials: S
authors.lastname: Choi
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA; Department of Biology, California State University, Northridge, CA 91325, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kelber
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Xinning
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Strnadel
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Ken
authors.initials: K
authors.lastname: Fujimura
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Martina
authors.initials: M
authors.lastname: Pasillas
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA.
authors.forename: Judith
authors.initials: J
authors.lastname: Coppinger
authors.affiliation: Department of Pathology, University of California, La Jolla, San Diego, CA 92093, USA; Moores Cancer Center, University of California, La Jolla, San Diego, CA 92093, USA. Electronic address: rklemke@ucsd.edu.
authors.forename: Richard
authors.initials: R
authors.lastname: Klemke
mesh_terms: D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D013058:Mass Spectrometry; D051379:Mice; D040901:Proteomics / Q000379:methods*
keywords: Actin scaffold; Cytoskeleton; Focal adhesion; Proteomics