title: Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis.
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.
authors: Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan.
authors: Akihiko
authors: A
authors: Ito
authors: Takahiro
authors: T
authors: Mimae
authors: Ying-Shan-Zhu
authors: YS
authors: Yamamoto
authors: Man
authors: M
authors: Hagiyama
authors: Jun
authors: J
authors: Nakanishi
authors: Masaoki
authors: M
authors: Ito
authors: Yoichiroh
authors: Y
authors: Hosokawa
authors: Morihito
authors: M
authors: Okada
authors: Yoshinori
authors: Y
authors: Murakami
authors: Tadashi
authors: T
authors: Kondo
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D054018:Lasers, Excimer; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction; D040901:Proteomics; D011554:Pseudopodia / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D017931:DNA Primers