Methods for Pseudopodia Purification and Proteomic Analysis
Directional cell migration (chemotaxis) plays a central role in a wide spectrum of physiological and pathological processes, including embryo development, wounding healing, immunity, and cancer metastasis (1, 2). The process of chemotaxis is characterized by the sustained migration of cells in the direction of an increasing concentration of chemoattractant and/or ECM protein. Upon sensing the chemoattractant cells response with localized amplification of signals on the side facing the gradient (3-7). The spatial signal propagation facilitates reorganization of the actin-myosin cytoskeleton leading to extension of a dominant pseudopodium (PD) only in the direction of chemoattractant (7-10). While it is clear that localized signaling is critical for pseudopodium formation and chemotaxis, the molecular mechanisms that mediate this response remain poorly defined. To investigate mechanisms of pseudopodia formation, we recently described a novel approach to separate the PD and cell body (CB) compartments for large scale proteomic and phosphoproteomic analyses using chambers equipped with microporous filters (Fig. 1) (3, 7, 11). This in vitro system recapitulates physiological events associates with pseudopodial protrusion through small openings in the ECM and the vessel wall during immune cell intravasation and cancer cell metastasis (12, 13). The model system has been used to reveal important signaling pathways and novel proteins that mediate cell migration. This model, combined with the state-of-the-art proteomics and phosphoproteomics technology, will provide an effective approach to systematically analyze the proteins that differentially localized or phosphorylated in the front and the back of polarized migrating cells. In the following sections, we will describe in detail the protocols used to purify the PD and CB compartments for large-scale proteomic and phosphoproteomic analyses using mass spectrometry.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 985076
- Report Number(s):
- PNNL-SA-56168; 400412000; TRN: US201016%%1761
- Journal Information:
- Sciences STKE Signal Transduction Knowledge Environment, 2007(400):p14, Vol. 2007, Issue 400
- Country of Publication:
- United States
- Language:
- English
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