Article Figures & Data
Figures
- Figure 1.
Rap1 regulates focal adhesion formation. A, B16F1 cells were maintained in suspension (−) or plated on fibronectin (FN) or laminin (LAM). Cell lysates were assayed for activated Rap1-GTP and total Rap1. Right, Rap1-GTP levels relative to suspension cells. Columns, mean from three experiments; bars, SD. B, B16F1 cells stably transfected with the empty pIRM21-IRES-dsFP593 vector or derivatives encoding FLAG-Rap1V12 or FLAG-Rap1GAPII (see Supplementary Fig. S1) were plated on fibronectin for 4 h and then immunostained for β1 integrin, paxillin, or pFAK. Representative confocal images of the cell-ECM interface are shown. Scale bar, 20 μm. By setting the threshold for fluorescence detection such that only signals from protein clusters (putative adhesion complexes) were detected, the adhesion area per cell for each protein was determined. Columns, mean for 50 cells from three experiments; bars, SD. C, B16F1 cells that were transiently cotransfected with talin-GFP plus the empty pIRM21-IRES-dsFP593 vector or derivatives encoding FLAG-Rap1V12 or FLAG-Rap1GAPII were plated on fibronectin for 4 h and then imaged. Transfected cells were identified by GFP and dsFP593 (dsRed; inset) fluorescence. Adhesion area per cell for talin-GFP was determined as in B. D, the indicated tumor cell lines were transiently transfected with the indicated vectors and plated on fibronectin for 4 h. The cells were immunostained with anti-FLAG to identify transfected cells and with either a pan–β1 integrin antibody (murine K1735M1 cells) or the HUTS4 antibody, which recognizes only the active form of human β1 integrin (human A375 and MDA-MB-231 cells). Representative confocal images of the cell-ECM interface (see Supplementary Fig. S3) were used to determine the β1 integrin–containing adhesion area per cell, as in B. ***, P < 0.001; **, P < 0.01, compared with control cells.
- Figure 2.
Rap1 regulates adhesion and cytoskeletal dynamics, cell polarization, and tumor cell motility. A, vector control, Rap1V12, and Rap1GAPII B16F1 cells expressing paxillin-GFP were plated on fibronectin for 4 h and then imaged continuously for 10 min. Focal adhesions in the first frame (0 min) were pseudocolored green and those in the final frame (10 min) were pseudocolored red before superimposing the two images to identify focal adhesions that were newly formed (red), disassembled (green), or stable (merge = yellow) over the 10-min period. Focal adhesion turnover (% either disassembled or newly assembled) is graphed. Columns, mean for 10 to 15 representative cells from two experiments; bars, SD. B, the indicated cells coexpressing actin-GFP were plated on fibronectin for 4 h and then imaged continuously for 5 min. Top, representative still frames. Scale bars, 20 μm. Bottom, composite images of pseudocolored frames collected at the indicated times. Stable localization of actin-GFP over the 5-min period appears white. The percentage of the total actin-GFP in five equally divided regions of >25 cells from three experiments is graphed. C, vector control, FLAG-Rap1V12–expressing, FLAG-Rap1GAPII–expressing, and Myc-Rap1N17–expressing B16F1 cells were plated for 16 h on fibronectin (2.5 μg/cm2) that was overlaid with fluorescent microspheres. F-actin was visualized with AlexaFluor488-phalloidin (green). Transfected cells were identified by immunostaining for FLAG or Myc (red). Representative confocal images are shown. Scale bar, 100 μm. The maximum distance from the edge of a cell to the edge of the area cleared of fluorescent beads (white arrows) for >50 cells from three experiments is graphed. The horizontal bar is the mean. ***, P < 0.001, compared with vector control cells.
- Figure 3.
Modulating Rap1 activity alters melanoma growth and metastasis in vivo. A, stably transfected B16F1 cells were injected s.c. into mice. All injections generated palpable tumors, which were excised and weighed after 14 d. Representative images of the tumors are shown. Right, Rap1-GTP (top) and total Rap1 (bottom) in lysates from excised subcutaneous tumors. B, stably transfected B16F1 cells were injected i.v. After 21 d, lungs were removed and photographed, and visible colonies were counted. Horizontal bars represent the mean values. ***, P < 0.001, compared with vector control cells.
- Figure 4.
Modulating Rap1 activity impairs melanoma cell extravasation. A, for competitive lung arrest assays, equal numbers of fluorescently labeled vector control B16F1 cells (green) and either Rap1V12- or Rap1GAPII-expressing (red) cells were coinjected i.v. into mice. At the indicated times, lungs were removed, fixed, sectioned, and stained with DAPI to mark all nuclei. Representative sections are shown. The relative number of the different cell populations in 10 to 30 500-μm2 sections from four experiments is graphed. Columns, mean; bars, SE. B, to assess tumor cell adhesion to endothelial cells under flow, fluorescently labeled B16F1 cells (green) were perfused across a HUVEC monolayer (red) for 10 min in the presence or absence of a β1 integrin blocking antibody. Representative images of tumor cells adhering to the monolayers are shown. Scale bars, 100 μm. The number of adherent cells per field is graphed. Columns, mean for more than four fields in each of three experiments; bars, SD. ***, P < 0.001, compared with control cells. C, for extravasation assays, fluorescently labeled B16F1 cell populations (green) were injected i.v. together with fluorescent dextran (red) to label the lung microvasculature. After 4 h, lung sections were prepared and imaged as in A. Scale bar, 50 μm. Tumor cells that did not colocalize with the labeled dextran were considered to have extravasated. Yellow, colocalized with dextran within the microvasculature; green, not colocalized, outside the microvasculature. For each cell type, the percentage of imaged cells that had extravasated was determined. Columns, mean for three experiments; bars, SD. **, P < 0.01, compared with control cells. Because Rap1V12- and Rap1GAPII-expressing cells were poorly retained in the lungs, five times as many cells were injected (input) to visualize a sufficient number of cells. D, stably transfected CMFDA-labeled B16F1 cells were added to Transwell chambers in which the filters were coated with a collagen I gel. The lower chamber contained FCS and TGF-β as chemoattractants. After 20 h, invasion of the cells into the three-dimensional collagen ECM was imaged. Representative Z-axis images from one of three separate experiments with similar results are shown.
- Figure 5.
Modulating Rap1 activity inhibits tumor cell TEM and alters adhesion dynamics in tumor cells interacting with endothelial cells. A, fluorescently labeled B16F1 cells (green) were plated on monolayers of bEND.3 microvascular endothelial cells. After 4 to 24 h, the cells were stained for F-actin and imaged by confocal microscopy. Representative images of confocal slices of the “bottom” (0 μm; B) and “top” (T) of the endothelial cells (4–5 μm), as in the schematics, are shown. Scale bar, 20 μm. Cells that reached the bottom plane were scored as having undergone TEM. B, TEM by stably transfected B16F1 cells, as well as transiently transfected K1735M1, A375, and MDA-MB-231 cells, was analyzed as in A. The percentage of imaged cells (>100 per point) that underwent TEM is graphed. Columns, mean for three experiments; bars, SE. C, transiently transfected A375 and MDA-MB-231 cells were plated on bEND.3 mouse endothelial cell monolayers. The HUTS4 antibody was used to detect active human β1 integrins (green). Insets, anti-FLAG antibodies were used to detect transfected cells (blue). Arrowhead indicates transfected cell. Representative confocal images of the tumor cell–endothelial cell interface are shown. Scale bar, 10 μm. The area per cell occupied by clustered active β1 integrins at the cell-cell interface was quantified as in Fig. 1. Columns, mean for 30 cells; bars, SD. D, A375 and MDA-MB-231 cells transiently cotransfected with β1 integrin–GFP and the indicated vector were plated on endothelial cell monolayers. Transfected cells were identified by dsFP593 fluorescence. Regions containing integrin clusters were photobleached, and fluorescence recovery was recorded in real time (see Supplementary Fig. S5). The maximal fluorescence recovery was taken as the percentage of the β1 integrin–GFP that was mobile (n > 30 adhesions per point). The horizontal bar is the mean. ***, P < 0.001; **, P < 0.01; *, P < 0.05, compared with vector control cells.
- Figure 6.
Modulating Rap1 activity reduces the ability of melanoma cells to form protrusions that cross capillary walls in vivo. A, stably transfected B16F1 melanoma cells were labeled with CellTracker Green and injected into the femoral artery of mice along with AlexaFluor555-conjugated PECAM-1 antibodies to mark downstream capillaries (red). The cremaster muscle was exteriorized and imaged for 1 to 2 h after injection. Representative images of cells lodging in capillaries are shown. Scale bar, 50 μm. The higher-magnification insets show a vector control cell with a protrusion that crosses the capillary wall as well as Rap1V12 and Rap1GAPII cells without such protrusions. In the right panel, a vector control cell that was treated with trypsin for 30 min before injection is shown lodged in a capillary. B, actin-GFP–expressing vector control B16F1 cells were injected into the femoral artery along with labeled PECAM-1 antibodies to mark capillary walls (red). After 1 h, cells that lodged within capillaries were imaged continuously for 15 min. Large image, still frame from the start of the imaging period (0 min) showing actin-GFP within a protrusion that crossed the capillary wall. Small images, the same protrusion at 2-min intervals from 0 to 14 min. Scale bars, 10 μm. C, using images generated as in A, the percentage of B16F1 cells with pseudopodial protrusions that penetrated the capillary wall was determined (mean ± SD for three experiments in which >20 cells were imaged per point). **, P < 0.01, compared with vector control cells. D, model of how integrin-induced Rap1 activation and cycling contributes to the formation of protrusions that promote tumor cell extravasation and metastasis.
Additional Files
Supplementary Data, Freeman, et al.
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Volume 70, Issue 11
