Methods for pseudopodia purification and proteomic analysis.
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.
Wang
Yingchun
Y
Department of Pathology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
Ding
Shi-Jian
SJ
Wang
Wei
W
Yang
Feng
F
Jacobs
Jon M
JM
Camp
David
D
Smith
Richard D
RD
Klemke
Richard L
RL
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002465:Cell Movement / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D002630:Chemotactic Factors / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D002630:Chemotactic Factors; D020543:Proteome; D014357:Trypsin