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Dock180 and ELMO1 Proteins Cooperate to Promote Evolutionarily Conserved Rac-dependent Cell Migration

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m307087200

ISSN

1083-351X

Autores

Cynthia Grimsley, Jason M. Kinchen, Annie‐Carole Tosello‐Trampont, Enrico Brugnera, Lisa B. Haney, Mingjian Lu, Qi Chen, Doris Klingele, Michael O. Hengartner, Kodi S. Ravichandran,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180·ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO·Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration. Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180·ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO·Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration. Cell migration is essential for many normal and abnormal biological processes including embryonic development, wound healing, the immune response, and tumor cell metastasis. In virtually every cell type examined, cell movement requires the activity of Rac, a member of the Rho family of small GTPases (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3768) Google Scholar, 2Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. 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Cell Sci. 2001; 114: 3795-3803Crossref PubMed Google Scholar). Rac can also mediate the assembly of multi-molecular signaling and adhesion complexes associated with these leading edge protrusions (9Webb D.J. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2002; 4: E97-E100Crossref PubMed Scopus (594) Google Scholar, 10Horwitz A.R. Parsons J.T. Science. 1999; 286: 1102-1103Crossref PubMed Scopus (341) Google Scholar). Despite their importance, the upstream signaling mechanisms that facilitate Rac activation during migration are not fully understood (11Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar, 12Symons M. Settleman J. Trends Cell Biol. 2000; 10: 415-419Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Recent work (13Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (145) Google Scholar, 14Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (335) Google Scholar, 15Reif K. Cyster J. Trends Cell Biol. 2002; 12: 368-373Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) has revealed an evolutionarily conserved protein superfamily with homology to Dock180 comprised of at least 11 mammalian members. Dock180, the prototype member of this superfamily, forms a basal complex with ELMO1 and together this complex functions as an unconventional two-part guanine nucleotide exchange factor (GEF) 1The abbreviations used are: GEFguanine nucleotide exchange factorDTCdistal tip cellELMOengulfment and cell motilityGFPgreen fluorescent proteinSHSrc homologyCMVcytomegalovirusHAhemagglutininGSTglutathione S-transferasePHpleckstrin homologyCRIBCdc42/Rac interactive binding.1The abbreviations used are: GEFguanine nucleotide exchange factorDTCdistal tip cellELMOengulfment and cell motilityGFPgreen fluorescent proteinSHSrc homologyCMVcytomegalovirusHAhemagglutininGSTglutathione S-transferasePHpleckstrin homologyCRIBCdc42/Rac interactive binding. specific for Rac (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). As with other GTPases, Rac functions as a binary switch by cycling between an inactive GDP-bound form and an active GTP-bound form. GEFs promote the exchange of GDP for GTP by stimulating the dissociation of GDP and stabilizing the nucleotide-free form, thereby facilitating association of GTP (17Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 18Worthylake D.K. Rossman K.L. Sondek J. Nature. 2000; 408: 682-688Crossref PubMed Scopus (303) Google Scholar). However, neither Dock180 nor ELMO1 contains an obvious Dbl homology domain, which is present in most other known mammalian GEFs for Rho family GTPases (19Hasegawa H. Kiyokawa E. Tanaka S. Nagashima K. Gotoh N. Shibuya M. Kurata T. Matsuda M. Mol. Cell. Biol. 1996; 16: 1770-1776Crossref PubMed Scopus (289) Google Scholar, 20Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Instead, Dock180 and its homologues contain a Docker domain that can interact directly with nucleotide-free Rac and mediate Rac GDP/GTP exchange in vitro (14Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (335) Google Scholar, 16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). guanine nucleotide exchange factor distal tip cell engulfment and cell motility green fluorescent protein Src homology cytomegalovirus hemagglutinin glutathione S-transferase pleckstrin homology Cdc42/Rac interactive binding. guanine nucleotide exchange factor distal tip cell engulfment and cell motility green fluorescent protein Src homology cytomegalovirus hemagglutinin glutathione S-transferase pleckstrin homology Cdc42/Rac interactive binding. In intact cells, however, the Docker domain alone is insufficient for efficient Rac GTP-loading and an interaction between Dock180 and ELMO1 is required for GEF activity (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). Moreover, Dock180 and ELMO1 functionally cooperate to promote phagocytosis of apoptotic cells, a Rac-dependent process that involves dynamic reorganization of the actin cytoskeleton (for review see Ref. 21Henson P.M. Bratton D.L. Fadok V.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 627-633Crossref PubMed Scopus (287) Google Scholar). Neither Dock180 nor ELMO1 alone can promote phagocytosis. Interestingly, these two proteins also colocalize on membrane ruffles (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar), which are actin-rich protrusions associated with the leading edge of migrating cells. One other member of this superfamily, termed Dock9 or Zizimin, was found to bind and specifically activate Cdc42 (another Rho family member) rather than Rac (13Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (145) Google Scholar). Thus, it is likely that the other members of this superfamily also function as GEFs. The biological roles of this new superfamily and the cellular contexts in which they function are important areas of continuing investigation (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3768) Google Scholar, 15Reif K. Cyster J. Trends Cell Biol. 2002; 12: 368-373Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Genetic studies in Drosophila and Caenorhabditis elegans have suggested that highly conserved homologues of Dock180 and ELMO1 function as critical upstream regulators of Rac. In Drosophila, mutations in the gene encoding Myoblast City (Dock180), which acts upstream of Drac1 (Rac), lead to defects in myoblast fusion, dorsal closure, and border cell migration (22Duchek P. Somogyi K. Jekely G. Beccari S. Rorth P. Cell. 2001; 107: 17-26Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 23Erickson M.R. Galletta B.J. Abmayr S.M. J. Cell Biol. 1997; 138: 589-603Crossref PubMed Scopus (230) Google Scholar, 24Nolan K.M. Barrett K. Lu Y. Hu K.Q. Vincent S. Settleman J. Genes Dev. 1998; 12: 3337-3342Crossref PubMed Scopus (179) Google Scholar, 25Rushton E. Drysdale R. Abmayr S.M. Michelson A.M. Bate M. Development. 1995; 121: 1979-1988PubMed Google Scholar). Worms deficient in either CED-5 (Dock180) or CED-12 (ELMO1) display defects in engulfment of apoptotic cells, axonal pathfinding, and migration of the distal tip cells (DTCs) (20Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 26Wu Y.C. Horvitz H.R. Nature. 1998; 392: 501-504Crossref PubMed Scopus (136) Google Scholar, 27Wu Y.C. Tsai M.C. Cheng L.C. Chou C.J. Weng N.Y. Dev. Cell. 2001; 1: 491-502Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 28Zhou Z. Caron E. Hartwieg E. Hall A. Horvitz H.R. Dev. Cell. 2001; 1: 477-489Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 29Reddien P.W. Horvitz H.R. Nat. Cell Biol. 2000; 2: 131-136Crossref PubMed Scopus (329) Google Scholar, 30Lundquist E.A. Reddien P.W. Hartwieg E. Horvitz H.R. Bargmann C.I. Development. 2001; 128: 4475-4488Crossref PubMed Google Scholar). Genetically, CED-5 and CED-12 were shown to function at the same step upstream of CED-10 (Rac) in these processes. It remains unknown, however, whether the Dock180 and ELMO1 proteins also regulate Rac-dependent mammalian cell migration and which structural features of these proteins are involved. Here, we provide evidence that Dock180 and ELMO1 functionally synergize to promote Rac-dependent cell migration using an in vitro Transwell migration assay. We also confirm these observations at an organismal level by rescue of CED-5 deficient worms with mutants of Dock180. Interestingly, based on studies using ELMO1 mutants, generalized Rac activation in cells alone is not sufficient to enhance migration but rather targeting via the N terminus of ELMO1 appears to be critical for Dock180·ELMO1-mediated migration. Plasmids and Antibodies—GFP-tagged ELMO1, ELMO1-Δ330-GFP and the ELMO1-FLAG-CAAX have been described previously (20Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). ELMO1-Δ531-FLAG-CAAX was generated by replacing the coding region for full-length ELMO1 in ELMO1-FLAG-CAAX with the coding region for residues 532-727. GFP-tagged T707, 6M, and Δ531 mutants of ELMO1 and the ΔSH3 mutant of Dock180 were generated by a PCR-based approach and were sequenced to confirm the appropriate mutations. The mutations in the 6M mutant are as follows: L689A, M691S, E692D, R696K, L697A, and L698A. Full-length Dock180, the DOHRS mutant plasmid, Dock180-CAAX, and FLAG-tagged Dock2 were provided by Dr. Matsuda (19Hasegawa H. Kiyokawa E. Tanaka S. Nagashima K. Gotoh N. Shibuya M. Kurata T. Matsuda M. Mol. Cell. Biol. 1996; 16: 1770-1776Crossref PubMed Scopus (289) Google Scholar, 32Kiyokawa E. Hashimoto Y. Kurata T. Sugimura H. Matsuda M. J. Biol. Chem. 1998; 273: 24479-24484Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 38Nishihara H. Kobayashi S. Hashimoto Y. Ohba F. Mochizuki N. Kurata T. Nagashima K. Matsuda M. Biochim. Biophys. Acta. 1999; 1452: 179-187Crossref PubMed Scopus (75) Google Scholar). The Δ357 and Dock-ISP mutants of Dock180 and the ELMO1-T625 mutant have been described previously (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). The plasmid-encoding Tiam1 (C1199) was provided by Dr. John Collard (Netherlands Cancer Institute) (48Sander E.E. ten Klooster J.P. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (729) Google Scholar). The pGL3-CMV-luciferase plasmid was obtained from Dr. Michael Smith (University of Virginia). A Peft-3 expression construct (20Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) was modified with NcoI-NotI sites in which FLAG-tagged DOCK180(wild type), DOCK180(ISP→AAA), or DOCK180-(358-1865) was cloned for subsequent expression in C. elegans. HA-tagged Dock3 was kindly provided by David Schubert (Salk Institute). The FLAG-tagged Rac1Q61L and Rac1T17N plasmids were obtained from Dr. Tom Parsons (University of Virginia). Purified rabbit polyclonal anti-ELMO1 has been described previously (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). Mouse monoclonal anti-GFP (clone B2), mouse monoclonal anti-HA (clone F7), goat polyclonal anti-Dock180 (clones N19 and C19), rabbit polyclonal anti-GST (clone Z5), and horseradish peroxidase-conjugated donkey anti-goat IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-FLAG (clone M2) was from Sigma. Mouse monoclonal anti-Rac (clone 23A8) was from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Amersham Biosciences. All of the immunoblots were developed using enhanced chemiluminescence (Pierce). Cell Culture and Transfection—293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% penicillin/streptomycin/glutamine. LR73 cells were maintained in Alpha's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin/glutamine. 293T cells were transiently transfected by the calcium phosphate method, and LR73 cells were transiently transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instruction. In all of the experiments, carrier DNA was added to keep equal plasmid concentration between different samples. Migration and Adhesion Assay—LR73 cells at ∼70% confluency were transiently transfected with pGL3-CMV-luciferase as a reporter construct in addition to the indicated plasmids in a 6-well plate. After 20 h, cells were harvested using trypsin-EDTA and resuspended in Opti-MEM medium supplemented with 2% fetal bovine serum. 1 × 105 cells were added in duplicate to the upper chamber of an untreated polycarbonate Transwell filter with 8-μm pores (Costar). Opti-MEM medium supplemented with 2% fetal bovine serum was also added to the lower chamber. After a 6-h incubation, Transwell filters were removed and the number of cells migrating completely through the filter to the lower chamber was assessed by quantitation of luciferase activity (Promega). 1 × 105 cells were also separately added in parallel to wells without Transwell filters for estimating total luciferase activity, upon which the percent migration was estimated for each transfection condition. The percent migration of control cells transfected with luciferase alone was set at 100%. The percentage of cells remaining attached to the filter underside was assessed by mechanically removing cells from the top of the filter with a cotton swab and determining the luciferase counts of the remaining cells. An aliquot of cells from each transfection condition was analyzed for expression of transfected proteins by immunoblotting. To examine adhesion under the migration assay conditions, cells were transfected, harvested, and resuspended as described above for the migration assay. 1 × 105 cells were then either plated in duplicate on the same 24-well Transwell filter placed in a 12-well plate (so that the filter lies flush with the bottom of the well, eliminating the bottom chamber) or plated in a separate well without a Transwell filter to estimate the total luciferase counts for each condition. At the indicated time points, the filters were then gently washed with phosphate-buffered saline and the percentage of transfected cells remaining attached to the top of the filter was determined with a luciferase assay. Scoring of C. elegans DTC Migration—The indicated Dock180 and CED-5-coding sequences were subcloned into the transgenic vector driven by the Peft-3 promoter. To create DOCK180-transgenic lines, worms were injected with test DNA at a concentration of 10 ng/μl along with Plim-7::GFP as described previously (52Mello C.C. Kramer J.M. Stinchcomb D.T. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2392) Google Scholar). Injected hermaphrodites were picked and allowed to have progeny. Transgenic progeny (that expressed Plim-7::GFP) were moved to individual plates and allowed to grow. Worms that transmitted the array were kept and assayed for expression (brightness of GFP signal) and transmittance of the array. Strains with the highest transmittance/GFP signal were kept for further analysis. Worms were maintained at 20 °C as described previously (53Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). Clean transgenic worms were moved to a large plate that was seeded with OP50 bacteria and allowed to propagate one generation. Worms were then scored under a Zeiss M2Bio-dissecting microscope equipped with epifluorescence. Worms with a gonad that deviated from the standard U-shaped tube was scored as migration defective. Only worms in which both the anterior and posterior arms were clearly visible were scored. Immunoprecipitations and Immunoblotting—Lysis, immunoprecipitation, and immunoblotting were performed as described previously (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar, 20Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). 293T cells were transiently transfected with 10 μg of Dock180 or Dock180 homologues and 3 μg of ELMO1 plasmids. For FLAG immunoprecipitations, cells were harvested and lysed 36 h after transfection and immunoprecipitated using anti-FLAG antibody directly coupled to Sepharose. For ELMO1 immunoprecipitations, anti-ELMO1 antibody was incubated with protein A-Sepharose (Santa Cruz Biotechnology Inc.) for 1 h and washed. Cells were then harvested and lysed 24 h after transfection and incubated with the beads for 1 h. Precipitated proteins were then assessed by SDS-PAGE and immunoblotting (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar). Rac GTP-loading Assay—Bacterially produced GST-CRIB proteins bound to glutathione-Sepharose beads were incubated with lysates of LR73 or 293T cells (transfected with the indicated plasmids) for 1 h at 4 °C. The beads were then washed, and the levels of Rac-GTP present in the lysates were analyzed by SDS-PAGE and immunoblotting for Rac. Phagocytosis Assay—LR73 cells were transiently transfected in duplicate with the indicated plasmids (either with GFP or fused to GFP) in a 24-well plate. 20 h after transfection, the cells were incubated with 2 μm of carboxylate-modified red fluorescent beads in serum-free medium (Sigma). After 2 h, the wells were then washed twice with cold phosphate-buffered saline, trypsinized, resuspended in cold medium (with 0.5% sodium azide), and analyzed by two-color flow cytometry. The transfected cells were recognized by their GFP fluorescence. Forward and side-scatter parameters were used to distinguish unbound beads from cells. For each point, 30,000 events were collected and the data were analyzed using Cell Quest software. As shown previously (31Tosello-Trampont A.C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), the majority of double-positive cells scored in the fluorescence-activated cell sorter assay represents particles engulfed by transfected cells or particles in the process of engulfment and do not represent beads simply bound to the cell surface. Microscopy—The indicated plasmids were transiently transfected into LR73 cells plated on Labtek chamber slides using LipofectAMINE 2000 reagent at the following concentrations: ELMO1-GFP or Δ531-GFP (1.0 μg) and Dock180 (1.5 μg). At 24 h post-transfection, the cells were fixed in 3.7% paraformaldehyde and permeabilized with phosphate-buffered saline, 0.1% Triton X-100, 0.1% bovine serum albumin. Cells were then stained with Alexa Fluor-568 phalloidin (Molecular Probes, Eugene, OR) for 20 min at room temperature and analyzed by confocal microscopy. The regions of overlay of the green ELMO1-GFP fluorescence with the red fluorescence of Alexa Fluor-568 phalloidin are represented in yellow. The images shown are representative of multiple cells with similar phenotypes from three independent experiments. To quantitate morphology, cells were classified as 1-rounded appearance, 2-spread but not polarized, or 3-polarized with visible leading and tail edges using confocal and epifluorescence analyses. The expression of Dock180 in a duplicate chamber was confirmed by direct immunostaining for Dock180 (data not shown). Before the Dock180 staining, the permeabilized cells were blocked with phosphate-buffered saline containing 0.1% bovine serum albumin and 10% normal donkey serum for 20 min at room temperature. Cells were then stained with a goat polyclonal anti-Dock180 antibody (1:40) for 30 min at 4 °C followed by a Texas Red-labeled donkey anti-goat antibody (1:40) for 30 min at 4 °C to visualize expression of Dock180. Cells were mounted using Vectashield-mounting medium (Vector Laboratories, Inc., Burlingame, CA 94010). An Olympus Fluoview BX50WI laser-scanning microscope with a ×60 LumPlanFI lens with an aperture of 2 (zoom ×1.5) was used to obtain images. The acquisition software was Fluoview FV200, version 3.3, and images were processed as entire pictures using Adobe PhotoShop, version 6.0. Dock180 and ELMO1 Coexpression Promotes Mammalian Cell Migration—To probe the role of Dock180 and ELMO1 proteins in mammalian cell migration, we developed a Transwell migration assay with LR73 cells. This is a variant of the Chinese hamster ovary cell line commonly used for cell migration studies and has also been used previously to investigate the role of Dock180 and ELMO1 in phagocytosis of apoptotic cells (16Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (464) Google Scholar, 31Tosello-Trampont A.C. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). During the development of this migration assay, we found that LR73 cells completely migrated to the bottom chamber when placed on an uncoated Transwell filter and did not move to the underside of the filter. To score the movement of transfected cells, we used a cotransfected luciferase reporter gene, which provided a convenient and quantitative readout for the simultaneous marking of transfected cells and scoring of their motility. In a 6-h migration assay, coexpression of wild type Dock180 and ELMO1 strongly promoted migration compared with control cells expressing luciferase alone (generally 4-6-fold in >20 independent experiments) (Fig. 1A). Under similar conditions, expression of Dock180 alone or ELMO1 alone did not promote migration, indicating a requirement for both proteins for this effect. Immunoblotting an aliquot of cells from the same experiment also confirmed that Dock180 and ELMO1 were comparably expressed under the different transfection conditions. It is noteworthy that an equal number of cells from each transfection condition were also plated in a separate chamber (without a filter) for estimating the total luciferase activity in each transfected population. By analyzing these luciferase counts, we confirmed that the luciferase expression, driven by a constitutive CMV promoter, was unaffected by cotransfection with either the Dock180 or ELMO1 plasmids (Fig. 1B). Furthermore, no significant difference in luciferase expression was detected in any of the transfection conditions reported in this paper. In addition, we also found that there was no increase in cell attachment to the underside of the Transwell filter in any of the transfection conditions (Fig. 1B). Because an alternate explanation for the observed enhancement of motility with Dock180·ELMO1 coexpression could be differences in the ability of these cells to attach to the Transwell filter, we examined this possibility more closely under similar conditions. Cells were independently plated from the different transfection conditions directly on an isolated filter (without a bottom chamber), after which their relative adherence was measured. Under these conditions, we found comparable attachment of cells transfected with Dock180 alone, ELMO1 alone, or Dock180·ELMO1 in the 6-h time frame of the migration assay (Fig. 1C). We also detected no significant differences among the various transfected samples in filter adhesion at 30 min and at 1 and 3 h (data not shown). This finding suggests that the enhanced migration due to Dock180·ELMO1 coexpression does not result from overall differences in the ability of the cells to adhere to the filter. We next examined whether the enhanced migration due to Dock180·ELMO1 coexpression was dependent on Rac activity. Cotransfection of a dominant negative form of Rac (RacT17N) inhibited the Dock180·ELMO1-dependent increased migration (Fig. 1A), suggesting that the enhancement of migration with Dock180·ELMO1 coexpression depends on Rac activation. ELMO1- and Rac-binding Regions of Dock180 Are Required for Migration—It has previously been determined that ELMO1 binding requires the N-terminal 357 amino acids of Dock180, whereas Rac binding occurs via the Docker domain (amino acids 1111-1657

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