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GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration

Ri-ichiroh Manabe*,{ddagger}, Mykola Kovalenko{ddagger}, Donna J. Webb§ and Alan Rick Horwitz

Department of Cell Biology, University of Virginia, Charlottesville VA 22908
* Present address: Ri-chiroh Manabe, Japan Science and Technology Corporation, Sekiguchi Biomatrix Signaling Project, c/o Aichi Medical University, 21 Karimata-Yasago, Nagakute, Aichi 480-1195, Japan
{ddagger} These authors contributed equally to this work



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  		Fig. 1. Subcellular localization of GIT1. (A) Schematic representation of the GIT1 mutants used in this study. (B) REF 52 cells were plated on fibronectin, fixed and stained for endogenous paxillin. (C) GFP fluorescence localized to adhesion-like structures in REF52 cells transfected with GFP-cGIT1. (D) CHO K1 cells were stained for endogenous GIT1. The arrows indicate localization to the cytoplasmic complexes. CHO K1 cells expressing (E) wild-type GFP-GIT1, (F) GFP-nGIT1, and (G) GFP-mini-GIT1 were viewed using fluorescence. Wild-type GIT1, cGIT1, and mini-GIT1, but not nGIT1, localized in adhesions, indicating that the adhesion-targeting domain resides at the C-terminus of GIT1. The localization of the wild-type GIT1 to the leading edge of a lamellipodium is indicated by an arrow in panel (E). WI-38 cells were antibody stained for (H) endogenous GIT1 and (I) paxillin. GIT1 colocalized with paxillin in adhesions. Bar, 10 µm.

 


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  		Fig. 2. GIT1 cytoplasmic complexes do not colocalize with endosomal markers. CHO K1 cells expressing medium levels of GFP-GIT1 were fixed, permeabilized and stained for transferrin receptor and mannosidase-2 (a Golgi marker). In separate experiments, GIT1-expressing CHO K1 cells were incubated with Cy-3 transferrin for 7 minutes or FM4-64 dye for 24 hours. Cells were viewed in fluorescence using confocal microscopy. GIT1 did not co-localize with any of these markers. Bar, 10 µm.

 


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  		Fig. 3. GIT1 cytoplasmic complexes contain paxillin and PAK. GFP-GIT1-expressing CHOK1 cells were either co-stained for paxillin or co-transfected with Myc-PAK and stained for Myc. (A) GFP-GIT1 co-localized with (B) Myc-tagged PAK in the cytoplasmic structures (arrows). In GFP-GIT1-expressing cells co-transfected with a PIX-binding-deficient mutant of PAK, (C) GIT1 localized in cytoplasmic complexes (arrows) but (D) the mutant PAK was not detected in these structures. Both (E) GFP-GIT1 and (G) endogenous GIT1 co-localized in cytoplasmic complexes with (F,H) endogenous paxillin as indicated by arrows. Bar, 10 µm.

 


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  		Fig. 4. GIT1 cytoplasmic complexes move into adhesions. CHOK1 cells stably expressing GFP-GIT1 were plated on 2 µg/ml fibronectin, and their movement captured using time-lapse fluorescence microscopy. (A) Cytoplasmic complexes containing GIT1 (two arrows show the path of two separate complexes) move toward a pre-existing adhesion and appear to fuse with it. (B,C) GFP-GIT1 (upper panels) complexes in adhesions co-localized with (B) paxillin and (C) vinculin (lower panels) in stably expressing CHOK1 cells. Bars, 10 µm. See Movie 1 at jcs.biologists.org .

 


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  		Fig. 5. GIT complexes (the two arrows indicate two separate complexes) move from an adhesion at the periphery toward at the cell cortex as the cell edge retracted. Cells were plated on 2 µg/ml fibronectin and the movement captured by time-lapse fluorescence microscopy. Bar, 10 µm. See Movie 2 at jcs.biologists.org .

 


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  		Fig. 6. GIT1 cytoplasmic complexes move from the rear toward the cell center. GFP-GIT1-expressing CHOK1 cells were filmed as described in Fig. 5. GIT1 complexes moved from the retracting edge of the cell toward the perinuclear area (white arrows). In the top left panel, the path of a GIT1 complex was tracked (white line) and the arrows indicate the position of the complex at the beginning (white arrow) and end (gray arrow) of the observation period. Bar, 10 µm. See Movie 3 at jcs.biologists.org .

 


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  		Fig. 7. Wild-type and cGIT1 enhance migration and protrusion formation. CHO K1 cells expressing GFP alone, wild-type GIT1-GFP or GFP-cGIT1 were plated on 2 µg/ml fibronectin, and their migration was captured using time-lapse microscopy (10 minute intervals). (A) The fraction of cells migrating at various speeds was calculated for each construct. Both wild type and cGIT1 increased the migration speeds when compared to cells expressing GFP alone. (B) Protrusive activity was expressed as the net positive change in cell area over time (10 minute). Cells expressing (D) wild-type GIT1 and (E) cGIT1 appeared much more protrusive when compared to cells expressing (C) GFP alone. The arrows in (D) and (E) indicate protrusions. Cells expressing (F,G) GFP alone or (H,I) cGIT1-GFP were stained for endogenous GIT1. In GFP-expressing cells, endogenous GIT1 localized to adhesion-like structures as well as to cytoplasmic complexes, as indicated by arrows in (F). In cells expressing (I) GFP-cGIT1, which localized in adhesion-like structures and at the leading edge, (H) endogenous GIT1 was prominent in membrane ruffles at the leading edge, arrows. Bar, 10 µm.

 


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  		Fig. 8. GIT1 targets PAK to adhesions and the membrane at the leading edge of the lamellipodium. GFP-GIT1-expressing cells were co-transfected with (A) Myc tagged-kinase-dead or (C-E) constitutively active PAK. Although a cell expressing (B) GFP-GIT1 alone showed lamellipodia-like protrusions (arrows), they were significantly reduced when (A) kinase-dead PAK was co-expressed. (D) Constitutively active PAK localized prominently to adhesions and the leading edge (arrows) when co-expressed with (C) GIT1. (E) Cells were co-stained for vinculin to verify localization of adhesions. Enhanced localization of constitutively active PAK to adhesions and the leading edge was not observed when PAK was co-expressed with either (F-H) n-GIT1 or (I-K) mini-GIT1, indicating the need for both the C- terminus of GIT1 and its central PIX-binding domain. (F-H) GFP-nGIT1 and (I-K) GFP-mini-GIT1 expressing cells were co-stained for (G,J) constitutively active PAK and (H,K) vinculin. Bar, 20 µm.

 


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  		Fig. 9. Constitutively active Rac enhances GIT1 localization to adhesions and decreases its presence in the cytoplasmic complexes. GFP-GIT1 -expressing or non-transfected CHOK1 cells were transfected with Myc-tagged V12-Rac or N17-Rac, fixed and stained with an anti-Myc antibody. Cells in the bottom panels were co-stained for endogenous GIT1. V12-Rac induced strong localization of GFP-GIT1 (top two panels) and endogenous GIT1 (bottom two panels) in adhesions, dramatically diminishing its localization in cytoplasmic complexes. Unlike V12-Rac, N17-Rac (middle panels) did not have an effect. Bar, 20 µm.

 

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