Rac1 and Aurora A regulate MCAK to polarize microtubule growth in migrating endothelial cells

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 1 -
  MCAK tracks with growing EB3-labeled MT plus ends. Live-cell imaging of GFP-MCAK (left) and mApple-EB3 (middle) and overlay (right) reveals that MCAK (green) tracks with EB3 (red) on growing MT plus ends at the leading edge of wound-edge HUVECs. Images were acquired by time-lapse spinning disk confocal microscopy (Yokogawa CSU-X1 spinning disk Andor Technology; coupled to a TiE microscope Nikon). Frames were taken at 2-s intervals for 2 min using a 500-ms exposure for the 488-nm (green) and a 600-ms exposure for the 560-nm (red) excitation. Bar, 2 µm.
• Video 2 -
  Aurora A tracks with MCAK-labeled MT plus ends. Live-cell imaging of Em–Aurora A (left) and mCherry-MCAK (middle) and overlay (right) reveal that Em–Aurora A (green) tracks with a proportion of mCherry-MCAK (red) on growing MT plus ends at the leading edge of wound-edge HUVECs. In addition to growing MCAK-labeled MT plus ends, Em–Aurora A labeling is also visible on filaments behind the growing MCAK-labeled MT plus ends. Images were acquired by time-lapse spinning disk confocal microscopy (Yokogawa CSU-X1 spinning disk Andor Technology; coupled to a TiE microscope Nikon). Frames were taken at 2-s intervals for 2 min using a 500-ms exposure for the 488-nm (green) and a 600-ms exposure for the 560-nm (red) excitation. Bar, 2 µm.
• Video 3 -
  Aurora A association with MTs is enhanced by Rac1 activation. Live-cell imaging of Em–Aurora A (left) and mApple-Tubulin (middle) and overlay (right) reveal that Em–Aurora A (green) association with MTs (red) is enhanced at the leading edge of wound-edge HUVECs in response to CA-Rac1 (red). Images were acquired by time-lapse spinning disk confocal microscopy (Yokogawa CSU-X1 spinning disk Andor Technology; coupled to a TiE microscope Nikon). Frames were taken at 2-s intervals for 2 min using a 500-ms exposure for the 488-nm (green) and a 600-ms exposure for the 560-nm (red) excitation. Bar, 2 µm.
• Video 4 -
  Aurora A association with MTs is reduced by Rac1 inactivation. Live-cell imaging of Em–Aurora A (left) and mApple-Tubulin (middle) and overlay (right) reveal that Em–Aurora A (green) association with MTs (red) is reduced at the leading edge of wound-edge HUVECs in response to DN-Rac1 (red). Images were acquired by time-lapse spinning disk confocal microscopy (Yokogawa CSU-X1 spinning disk Andor Technology; coupled to a TiE microscope Nikon). Frames were taken at 2-s intervals for 2 min using a 500-ms exposure for the 488-nm (green) and a 600-ms exposure for the 560-nm (red) excitation. Bar, 2 µm.