Fig. 4: Selective association of T-Plastin with extending but not retracting protrusions.

a HUVEC expressing T-Plastin-mRuby3 and F-tractin-mCitrine were plated sparsely and imaged over time. The ratio of T-Plastin over F-tractin is shown as a parula colormap with yellow and blue indicating high (99th percentile) or low (3rd percentile) enrichment of T-Plastin, respectively and is the same scale in all panels using the parula colormap. The cell edge from each previous timepoint image is shown as a red outline; protrusions are present outside the red line while retractions lie within. Subregions blown up for clarity are denoted with a white asterisk. b Regions from of the cell edge from (a) were divided into windows for kymographic analysis of cell edge velocity and T-Plastin enrichment. Arrows depict edge velocities for each window between frames. c Kymographs of window edge velocities with protrusions shown in red (positive, +0.65 µm/s) and retractions in blue (negative, −0.65 µm/s). Parula kymograph shows the corresponding ratio of T-Plastin over F-tractin at same scale as (a) in the same windows analyzed for edge velocity. Dotted box shows region blown up in (d). d Region from (c) with protrusion areas highlighted in black and corresponding regions of T-Plastin enrichment also highlighted. e Temporal cross-correlation analysis of protrusion events shown in (a–d). Cross-correlation coefficients of T-Plastin (purple) and F-tractin (green) are shown relative the incidence of protrusion at 0 min. f Similar to (a), except cells also expressed MYL9-mTurq to mark myosin activity. Cells were treated with CK666 where indicated. g Similar to (f), except cells were first treated with 300 nM Jasplakinolide then CK666. Bars, 5 µm.