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

Akihiko Ito, Takahiro Mimae, Ying Shan Zhu Yamamoto, Man Hagiyama, Jun Nakanishi, Masaoki Ito, Yoichiroh Hosokawa, Morihito Okada, Yoshinori Murakami, Tadashi Kondo

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

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 <0.05). 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.

Original languageEnglish
Pages (from-to)1374-1385
Number of pages12
JournalLaboratory Investigation
Volume92
Issue number9
DOIs
Publication statusPublished - 2012 Sep
Externally publishedYes

Keywords

  • excimer laser ablation
  • mass spectrometry
  • proteome
  • pseudopodia
  • two-dimensional difference gel electrophoresis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

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