Fig. 3

Kinematics of CDRs. a CDRs formation anticipates protrusion extension with a typical lag phase. Top: Still images from a representative time-lapse (Supplementary Movie 3) of MEFs stimulated by PDGF delivered with a micropipette. Grey arrow indicates the position of released PDGF; red and white arrows indicate CDR and protrusions extension, respectively. The white dashed line indicates the ROI used to perform the kymograph (bottom right panel). Scale bar, 20 µm. Bottom left: Cartoon depicting the dynamics of CDRs and protrusions. Bottom right: kymograph of leading-edge dynamics. T₀ PDGF stimulation time, T₁ CDR closure time, T₂ protrusion extension times. b RAB35 promotes the formation of multiple CDRs that expand centrifugally and precede the extension of leading-edge protrusions. Top: pSLIK-HA-RAB35-human-MEFs were infected with EGFP-LifeAct and treated or not with doxycycline to induce transgene expression. Samples were monitored by time-lapse every 30″ for 60′ (Supplementary Movie 4) and still images are shown. Scale bar, 50 µm. Boxes indicate magnified areas shown on the side (Scale bar, 20 µm). Red and white arrows indicate CDRs and protrusion edge, respectively. Bottom left: the lag phase between CDR closure and the subsequent protrusion extension is expressed as mean ± SD (n = 30 cells/experiments, three independent experiments). Bottom right: RAB35 expression was verified by imunoblotting. c Travelling CDR waves induced by RAB35. Top: pSLIK-HA-RAB35-human-MEFs were infected with EGFP-LifeAct and treated or not with doxycycline to induce transgene expression. Samples were monitored by time-lapse every 30″ for 20′. Still images from time-lapse sequence (Supplementary Movie 5). Scale bar, 50 µm. The dashed line indicates the ROI used to perform the kymograph. Middle: Kymograph of cell edge (blue) and CDR (red). Bottom: The propagation speed of CDR (red) and leading-edge protrusions (blue) induced by RAB35 is expressed as mean ± SD (n = 30 cells, three independent experiments). d Oscillating CDR waves induced by RAB35. Doxycycline-treated pSLIK-HA-RAB35-human-EGFP-LifeAct-expressing MEFs were imaged every 30” for 1 h period. Still fluorescence images from time-lapse sequence (Supplementary Movie 6). Red arrows point the recurrent CDR formation. Scale bar, 10 µm. A representative frequency of recurrent waves. Red arrows indicate peaks that correspond to CDRs