Supplementary Figure 1: Overexpression of 3 or CD151 does not promote stress fibre formation.

(A) Example confocal microscopy images of control cells plated on LN and fixed and stained for endogenous 3 integrin and CD151. (B) Cells co-expressing GFPa3 and mRFP-CD151 plated on LN were fixed and stained with phalloidin-633 to image F-actin. Cells were imaged by confocal microscopy and stress fibre area analysed as in Figure 3D. Data is pooled from >50 cells per condition over 2 independent experiments. Error bars are SEM. (C) Quantification of cumulative FRET efficiency data from experiments shown in Fig 2B but showing standard deviations of data sets. Data is pooled from >12 cells per condition over 3 independent experiments. Error bars are SD, *=p<0.01 compared to equivalent cells on COL.

Supplementary Figure 2: 3 and 5 integrins do not form direct complexes with CD63.

Cells co-transfected with 3-GFP or 5-GFP and CD63-mRFP plated on COL, LN or FN and subjected to analysis of FRET by FLIM. Example images are shown for each. Images show the GFP multiphoton intensity image and lifetime images mapping spatial FRET across the cells, which are depicted using a pseudocolour scale (blue = normal GFP lifetime, red = FRET). Quantification of cumulative FRET efficiency data from experiments shown in histogram. Data is pooled from >9 cells per condition over 3 independent experiments. Error bars are SEM.

Supplementary Figure 3: Knockdown of 3 does not lead to increased 21 association with CD151.

(A) FACS analysis of cell surface levels of specified integrin subunits in 3 integrin shRNA expressing cells represented as % relative to shCon cells. Data is pooled from 3 independent experiments. Error bars are SEM. (B) Example confocal images of control or 3 integrin shRNA expressing cells fixed and stained for endogenous 2 integrin (green), CD151 (red) and F-actin (phalloidin, blue). (C) Cells co-transfected with 2-GFP and CD151-mRFP plated on COL and subjected to analysis of FRET by FLIM. Example images are shown for each. Images show the GFP multiphoton intensity image, a widefield RFP acceptor image and lifetime images mapping spatial FRET across the cells, which are depicted using a pseudocolour scale (blue = normal GFP lifetime, red = FRET). Quantification of cumulative FRET efficiency data from experiments shown in histogram. Data is pooled from >11 cells per condition over 2 independent experiments. Error bars are SEM. (D) Quantification of RhoA activation using acceptor photobleaching FRET in control or 3-silenced cells plated on COL and treated with control (IgG) or integrin function blocking antibodies (10g/ml for 30 minutes). Data is pooled from >18 cells per condition over 2 independent experiments. Error bars are SEM. **=p<0.01 and ***=p<0.005 compared to respective controls.

Supplementary Figure 4: Suppression of active RhoA in control and 3 silenced cells.

(A) Quantification of CD151-CD151 FRET in control or 3-silenced cells plated on COL and treated with control (IgG) or integrin function blocking antibodies (10g/ml for 30 minutes). Data is pooled from >14 cells per condition over 2 independent experiments. Error bars are SEM. (B) Analysis of RhoA activation using acceptor photobleaching FRET in control cells expressing mRFP alone or CD151 QRD-mRFP. Data is pooled from >16 cells per condition over 2 independent experiments. Error bars are SEM. (C) Cells expressing RhoA FRET biosensor were treated with C3 (1M, 4h) or co-transfected with p190RhoGAP and subjected to analysis of active RhoA levels by acceptor photobleaching FRET. Graph of cumulative FRET efficiency pooled from >15 cells per condition over 2 independent experiments. Error bars are SEM. **=p<0.01 and ***=p<0.005 compared to respective controls.

Supplementary Movie 1: Localisation of 3 and CD151 in cells on COL

Confocal time-lapse movies of cells expressing3-GFP (green) and CD151-mRFP (red) migrating on COL. Stills from this movie are shown in Figure 1C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 2: Localisation of 3 and CD151 in cells on LN

Confocal time-lapse movies of cells expressing3-GFP (green) and CD151-mRFP (red) migrating on LN. Stills from this movie are shown in Figure 1C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 3: Localisation of 3 and CD151 in cells on FN

Confocal time-lapse movies of cells expressing3-GFP (green) and CD151-mRFP (red) migrating on FN. Stills from this movie are shown in Figure 1C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 4: Localisation of CD151 in shControl cells on COL

Confocal time-lapse movies of shCon cells expressing CD151-GFP and lifeact-mRFP migrating on COL. Stills from this movie are shown in Figure 3C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 5: Localisation of CD151 in sh3 cells on COL

Confocal time-lapse movies of sh3 cells expressing CD151-GFP and lifeact-mRFP migrating on COL. Stills from this movie are shown in Figure 3C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 6: Localisation of CD151 in shControl cells on LN

Confocal time-lapse movies of shCon cells expressing CD151-GFP and lifeact-mRFP migrating on LN. Stills from this movie are shown in Figure 3C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 7: Localisation of CD151 in sh3 cells on LN

Confocal time-lapse movies of sh3 cells expressing CD151-GFP and lifeact-mRFP migrating on LN. Stills from this movie are shown in Figure 3C. Movies were acquired as one image every 30 seconds. Playback rates of movies shown are 6 frames per second. Scale bars are 10m.

Supplementary Movie 8: Dynamics of Lifeact-GFP in shCon cells in 3D Cy3-labelled matrigel

3D reconstructions of ~20 z-stacks of confocal time-lapse movies through the central 8m plane of shCon cells expressing lifeact-GFP embedded in 3D matrigel spiked with 1:3 cy3-labelled laminin. Movies acquired over 60 minutes at 2 min intervals. Playback rate is 6 frames/sec. Scale bar (shown in first frame) is 10m.

Supplementary Movie 9: Dynamics of Lifeact-GFP in shCon cells in 3D Cy3-labelled collagenI

3D reconstructions of ~20 z-stacks of confocal time-lapse movies through the central 8m plane of shCon cells expressing lifeact-GFP embedded in 3D collagen spiked with 1:3 cy3-labelled collagen. Movies acquired over 60 minutes at 3 min intervals. Playback rate is 6 frames/sec. Scale bar (shown in first frame) is 10m.

Supplementary Movie 10: Dynamics of Lifeact-GFP in shCon cells in 3D matrigel

3D reconstructions of ~50 z-stacks of confocal time-lapse movies of shCon cells expressing lifeact-GFP embedded in 3D matrigel. Movies acquired over 21 minutes at 1 min intervals. Playback rate is 6 frames/sec. Scale bar (shown in first frame) is 10m.

Supplementary Movie 11: Dynamics of Lifeact-GFP in sh3 cells in 3D matrigel

3D reconstructions of ~50 z-stacks of confocal time-lapse movies of sh3 cells expressing lifeact-GFP embedded in 3D matrigel. Movies acquired over 21 minutes at 1 frame per min. Playback rate is 6 frames/sec. Scale bar (shown in first frame) is 10m.

Supplementary Movie 12: Dynamics of Lifeact-GFP in shCon cells in 3D collagen

3D reconstructions of ~50 z-stacks of confocal time-lapse movies of shCon cells expressing lifeact-GFP embedded in 3D collagen. Movies acquired over 21 minutes at 1 min intervals. Scale bar (shown in first frame) is 10m.

Supplementary Movie 13: Dynamics of Lifeact-GFP in sh3 cells in 3D collagen

3D reconstructions of ~50 z-stacks of confocal time-lapse movies of sh3 cells expressing lifeact-GFP embedded in 3D collagen. Movies acquired over 21 minutes at 1 frame per min. Scale bar (shown in first frame) is 10m.

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