A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front–back polarity

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 7 -
  Anterior localization of GFP–MHCII-B 1935D in CHO.K1 cells rescued with GFP–MHCII-B 1935D. MHCII-B–depleted CHO.K1 cells were transfected GFP–MHCII-B 1935D (green) and allowed to migrate on fibronectin. Phase-contrast images are shown on right. Confocal images were captured in a laser-scanning confocal microscope (SP5; Leica). Frames were taken every 9 s for 20 min.
• Video 1 -
  Loss of adhesion elongation in MHCII-A–depleted CHO.K1 cells. MHCII-A–depleted CHO.K1 cells were cotransfected with paxillin-mCherry (single channel) and allowed to migrate on fibronectin. TIRF images were captured in a TIRF microscope (IX70; Olympus) coupled to a CCD camera (Retiga EXi; QImaging). Frames were taken every 5 s for 12.5 min.
• Video 2 -
  Restoration of adhesion elongation in MHCII-A-depleted CHO.K1 cells rescued with wild type GFP–MHCII-A. MHCII-A–depleted CHO.K1 cells were cotransfected with paxillin-mCherry (left) and wild-type GFP–MHCII-A (right) and allowed to migrate on fibronectin. TIRF images were captured in a TIRF microscope (IX70; Olympus) coupled to a CCD camera (Retiga EXi; QImaging). Frames were taken every 5 s for 12.5 min.
• Video 5 -
  Loss of stable front–rear polarization in MHCII-B–depleted CHO.K1 cells rescued with GFP–MHCII-B 1935D. MHCII-B–depleted CHO.K1 cells were cotransfected with mCherry-vinculin (left) and GFP-MHCII-B 1935D (right) and allowed to migrate on fibronectin. TIRF images were captured in a TIRF microscope (IX83/TIRF Mitico; Olympus) coupled to an electron-multiplying CCD camera (ImagEM X2; Hamamatsu Photonics). Frames were taken every 30 s for 50 min.
• Video 6 -
  Localization of wild-type GFP–MHCII-B away from the protrusion in CHO.K1 cells rescued with wild-type GFP–MHCII-B. MHCII-B–depleted CHO.K1 cells were transfected wild-type GFP–MHCII-B (green) and allowed to migrate on fibronectin. Phase-contrast images are shown on right. Confocal images were captured in a laser-scanning confocal microscope (SP5; Leica). Frames were taken every 9 s for 20 min.
• Video 3 -
  Lack of restoration of adhesion elongation in MHCII-A–depleted cells rescued with GFP–MHCII-A+5S, which is positioned away from the protrusion. MHCII-A–depleted CHO.K1 cells were cotransfected with paxillin-mCherry (left) and GFP–MHCII-A+5S (right) and allowed to migrate on fibronectin. TIRF images were captured in a TIRF microscope (IX70; Olympus) coupled to a CCD camera (Retiga EXi; QImaging). Frames were taken every 5 s for 16.6 min.
• Video 4 -
  Stable front–rear polarization of MHCII-B–depleted CHO.K1 cells rescued with wild-type GFP–MHCII-B. MHCII-B–depleted CHO.K1 cells were cotransfected with mCherry-vinculin (left) and wild-type GFP–MHCII-B (right) and allowed to migrate on fibronectin. TIRF images were captured in a TIRF microscope (IX83/TIRF Mitico; Olympus) coupled to an electron-multiplying CCD camera (ImagEM X2; Hamamatsu Photonics). Frames were taken every 30 s for 50 min.