title: In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion.
abstract: Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.
authors.affiliation: Department of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501,  Japan.
authors.forename: Eishu
authors.initials: E
authors.lastname: Hirata
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yukinaga
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Arakawa
authors.forename: Susumu
authors.initials: S
authors.lastname: Miyamoto
authors.forename: Takaharu
authors.initials: T
authors.lastname: Okada
authors.forename: Erik
authors.initials: E
authors.lastname: Sahai
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D005909:Glioblastoma / Q000201:enzymology* / Q000473:pathology*; D020662:Guanine Nucleotide Exchange Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology / Q000517:prevention & control; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: C466321:DOCK9 protein, human; C579360:DOCK9 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein