Methods for Pseudopodia Purification and Proteomic Analysis
Yingchun Wang1,
Shi-Jian Ding2,
Wei Wang1,
Feng Yang3,
Jon M. Jacobs3,
David Camp, II3,
Richard D. Smith3, and
Richard L. Klemke*1
1Department of Pathology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
2Department of Pathology/Microbiology, University of Nebraska Medical Center, Omaha, NE 68198–6805, USA.
3Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Abstract:
Directional cell migration requires the formation of a dominant pseudopodium in the direction toward which the cell migrates. When a migratory cell is stimulated with a chemoattractant or extracellular matrix (ECM) gradient, it responds with localized amplification of signals on the side facing the gradient. The signals mediate reorganization of the actin-myosin cytoskeleton, leading to morphological polarization of the cell and pseudopodium extension. To identify these signals, we developed an approach to biochemically isolate the pseudopodium from the cell body using 3.0-micrometer porous filters for large-scale quantitative proteomic and phosphoproteomic analysis. Here, we detail the methodology for pseudopodium purification and proteomic analysis. This model system should be widely applicable for the analysis of the pseudopodium proteome from various migratory cell lines, including primary and cancer cell lines stimulated with a diverse array of chemoattractants, ECM proteins, or both.
*Corresponding author: Richard L. Klemke, Department of Pathology and Moores Cancer Center, University of California, San Diego, Basic Science Building 1040, 9500 Gilman Drive, MC 0612, La Jolla, CA 92093, USA. Telephone: (858) 822-5610; fax: (858) 822-4566; e-mail: rklemke{at}ucsd.edu
