Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie S1 - Supplementary Movie 1. Nck is required for the assembly of the complex actin network enabling the protrusive function of invadosomes. Three-dimensional rendering of representative stacks of SR-SIM images from control (shScr, top panels) and Nck-silenced (shNck, bottom panels) cells. Cells were stained for F-actin with fluorescent phalloidin and immunostained for endogenous cortactin with a specific antibody. The arrows indicate invadosomes,typically prominent in control cells, displaying cortactin accumulation at the tip (ventral-most focal planes) of comet-like actin structures.
- Movie S2 - Supplementary Movie 2.Loss of Nck decreases the ventral surface mobility and accumulation of MMP14 in breast carcinoma cells. Representative TIRF time-lapse series from control (shScr, left panels) and Nck-silenced (shNck, right panels) MDA-MB-231 cells expressing mCherry-MMP14. Images are presented using an intensity modulated display to associate intensity (surface localization, top panels) with color and hue (red = high activity; blue = low activity). Image thresholding was used to show isolated mCherry-MMP14 vesicles reaching the ventral surface of the cell (bottom panels).
- Movie S3 - Supplementary Movie 3. Representative time-lapse series of the FRET/CFP image ratio of control (shScr, top) and Nck-depleted (shNck, bottom) MDA-MB-231 cells expressing the Cdc42 FRET-based biosensor. Images were collected at 1 min intervals during ∼ 60 min. Warm and cool colors indicate high vs. low FRET efficiency (i.e. high vs. low activity). Notice the formation of a front-rear axis of polarity and high FRET efficiency localized mostly at the leading edge in the control cell. In contrast, the FRET signal is highly increased and delocalized (non-polarized) in Nck-depleted cells. Movies are played at a rate of 15 frames/sec.
- Movie S4 - Supplementary Movie 4. Representative time-lapse series of the FRET/CFP image ratio of control (shScr, top) and Nck-depleted (shNck, bottom) MDA-MB-231 cells expressing the RhoA FRET-based biosensor. Images were collected at 10 second intervals during ∼ 30 min. Warm and cool colors indicate high vs. low FRET efficiency (i.e. high vs. low activity). Notice the formation of a front-rear axis of polarity and high FRET efficiency localized prominently at the leading edge in the control cell. In contrast, the FRET signal is low and delocalized (non-polarized) in Nck-depleted cells. Movies are played at a rate of 30 frames/sec.