Methods for Pseudopodia Purification and Proteomic Analysis

Wang, Yingchun; Ding, Shi‐Jian; Wang, Wei; Yang, Feng; Jacobs, Jon; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

doi:10.1126/stke.4002007pl4

Cited by 23 publications

(28 citation statements)

References 22 publications

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“…Pseudopodia and cell bodies were purified from Cos-7 cells as described (9)(10)(11). PY proteins were purified by immunoprecipitation and identified by MudPIT and mass spectrometry (see SI Experimental Procedures for details).…”

Section: Methodsmentioning

confidence: 99%

“…S1A) (9). To identify kinases and their substrates that spatially regulate these processes, we used a strategy that allows for the large-scale purification of pseudopodia actively extending toward an LPA gradient (9)(10)(11). The relative differences in pY proteins from pseudopodia were compared with pY proteins isolated from purified cell bodies by using pY immunoaffinity purification followed by MudPIT (14).…”

Section: Proteomic and Bioinformatic Analyses Of The Pseudopodial Pymentioning

confidence: 99%

“…Therefore, understanding how this structure is regulated is crucial to understanding how cells migrate and invade tissues during cancer progression. To this end, we described a novel method for purifying the pseudopodia from cells for signal transduction and proteomic studies (9)(10)(11). Using this fractionation method and quantitative mass spectrometry (MS), we have profiled the relative differences in the pseudopodium and cell body proteomes (10).…”

mentioning

confidence: 99%

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Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Wang¹,

Kelber²,

Cao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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109

190

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Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Proteomic and Bioinformatic Analyses Of The Pseudopodial Pymentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Wang¹,

Kelber²,

Cao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

109

190

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“…After removing the fibronectin-containing D-PBS, the membrane was bottomed with a 1:1 ratio of culture medium and the above-mentioned NIH3T3-conditioned medium and the culture medium was added to the membrane. 6 Next, 1 Â of NIH3T3 cells/well were seeded in 12-well culture plate and cultured on the membrane for 2 days at 37 1C in a 5% CO 2 incubator.…”

Section: Cell Culture On Porous Membranementioning

confidence: 99%

“…Previously, mass spectrometry-based proteomics combined with this approach were used to identify numerous proteins in pseudopodia protrusions. [4][5][6] However, there were critical limitations to the method used in the previous report. Manual separation of pseudopodia from cell bodies created the potential for contamination of cell body components into the pseudopodial extract.…”

mentioning

confidence: 99%

Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis

Ito

Mimae

Yamamoto³

et al. 2012

Laboratory Investigation

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We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold differences and P-value of o0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. This method will provide insights into the molecular details of pseudopodia and a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies.

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A Boyden chamber‐based method for characterization of astrocyte protrusion localized RNA and protein

Thomsen

Nielsen

2011

Glia

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The Boyden chamber assay has been developed for various cell migration and invasion protocols. One variant of the Boyden chamber assay is the pseudopodium isolation assay, which has been developed to identify RNA and proteins localized in pseudopodia cell protrusions. Astrocytes are the most abundant cell type in the CNS and typically extend long cellular protrusions. Increasing interest emerges concerning for example the growth mechanisms and functions of astrocytes in respect to brain development, re-uptake of neurotransmitters in the synaptic cleft and glial scar formation. Protein and RNA localization mechanisms have been extensively examined in neurons and shown to play pivotal roles for the functional presence of specific protein components in neuronal protrusions. Here we present a simple Boyden chamber based method to isolate astrocyte cell protrusions for biochemical analysis. We have succeeded in isolating protrusion localized RNA and protein from the mouse astrocyte cell line, C8-S, and mouse primary astrocytes. This is exemplified in biochemical analyses showing specific localization of the mRNA for Ras-related protein (Rab13), Plakophilin-4 (Pkp4), Ankyrin Repeat Domain 25 (Ankrd25), and inositol polyphosphate-1-phosphatase (Inpp1) in the protrusions of both C8-S cells and primary astrocytes. Concordant, the Pkp4 protein was also predominantly localized in the protrusions of C8-S and primary astrocytes. The described methodology can be the basis for both genome wide and specific descriptive and functional studies of RNA and protein localization in the protrusions of astrocytes which could contribute considerably to the existing knowledge of astrocyte functions in the CNS.

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Methods for Pseudopodia Purification and Proteomic Analysis

Cited by 23 publications

References 22 publications

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis

A Boyden chamber‐based method for characterization of astrocyte protrusion localized RNA and protein

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