F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 2 -
  Time-lapse video of PI3K signaling and protrusion during photoactivation of Rac near filopodia, as depicted in Fig. 1 C. TIRF microscopy images of an NIH 3T3 cell coexpressing mCerulean-PA-Rac and mCherry-AktPH (pseudocolored) were acquired every minute for 79 min. Time stamp shows hours and minutes. The red spots indicate the timing and area of photoactivation. Bar, 20 µm.
• Video 6 -
  Time-lapse video showing adhesion formation and PI3K signaling during protrusion over actin bundles, as depicted in Fig. 4 B. TIRF microscopy images of an NIH 3T3 cell expressing mCherry-AktPH (left) and EGFP-paxillin (right) were acquired every 30 s for 10 min. Time is given in minutes and seconds. An overlay of the two channels is shown at right. Bar, 5 µm.
• Video 8 -
  Time-lapse video showing ablation of localized PI3K signaling by Arp2/3 inhibition in uninduced cells, but not in cells with tamoxifen-induced knockout of Arpc2, as depicted in Fig. 5 C. Fibroblasts with conditional knockout of the Arpc2 (p34) subunit of the Arp2/3 complex, transfected with FP-AktPH (pseudocolored), were either uninduced (top) or tamoxifen-induced (bottom). TIRF images were acquired every 30 s for 19 min in separate experiments. At the indicated time, the Arp2/3 complex was inhibited by addition of 100 µM CK666. Time is given in minutes and seconds. Bar, 20 µm.
• Video 4 -
  Time-lapse video of PI3K signaling and fascin localization during protrusion over an F-actin bundle, as depicted in Fig. 3 A. TIRF microscopy images of an NIH 3T3 cell coexpressing mCherry-AktPH (left, pseudocolored) and EGFP-fascin (right, inverted grayscale) were acquired every 30 s for 67 min. Time stamp is shown in hours, minutes, and seconds. Bar, 10 µm.
• Video 1 -
  Time-lapse video of filopodia directing nascent lamellipodia, as depicted in Fig. 1 A. TIRF microscopy images of an NIH 3T3 cell expressing EGFP-AktPH (pseudocolored) were acquired every 15 s for 132 min. Time stamp is shown in minutes, seconds, and milliseconds. Bar, 20 µm.
• Video 10 -
  Time-lapse video of multiple F-actin bundles directing protrusions that coalesce to form a broad lamellipod, as depicted in Fig. 6 A. TIRF microscopy images of an IA32 MEF coexpressing EGFP-fascin (inverted grayscale) and mCherry-AktPH (pseudocolored) were acquired every 30 s for 21 min. Time is given in minutes and seconds. Bar, 10 µm.
• Video 5 -
  Time-lapse video of adhesion dynamics during protrusion over F-actin bundles, as depicted in Fig. 4 A. TIRF microscopy images of an NIH 3T3 cell expressing mCherry-Fascin (left, grayscale) and EGFP-paxillin (center, pseudocolored) were acquired every 30 s for 20 min. Time is given in minutes and seconds. An overlay of the two channels is shown at right. Bar, 5 µm.
• Video 3 -
  Time-lapse video of F-actin bundles directing lamellipodial protrusion, as depicted in Fig. 2 A. TIRF microscopy images of an NIH 3T3 cell expressing EGFP-fascin (inverted grayscale) were acquired every 30 s for 21 min. Time stamp shows minutes and seconds. Bar, 10 µm.
• Video 9 -
  Time-lapse video of protrusion dilation waves, as depicted in Fig. 5 D. TIRF microscopy images were of an NIH 3T3 cell expressing mCherry-AktPH (pseudocolored) were acquired every 10 s for 30 min. Time is given in minutes and seconds. Bar, 10 µm.
• Video 7 -
  Time-lapse video showing ablation of localized PI3K signaling by FAK inhibition in cells plated on fibronectin but not on poly-lysine, as depicted in Fig. 4 C. TIRF microscopy images of NIH 3T3 cells expressing FP-AktPH (pseudocolored), plated on either fibronectin (left) or poly-lysine (right), were acquired every 30 s for 17 min in separate experiments. At the indicated time, FAK activity was inhibited by addition of 10 µM FAK inhibitor II. Time is given in minutes and seconds. Bar, 20 µm.