Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

Files in this Data Supplement:

• Supplemental Material (PDF)
• Video 1 -
  MV dynamics in confluent cells. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) of the apical cortex region of a confluent MDCK cell that has been stably transfected with Lifeact-GFP to label actin. Bar, 2 µm. Frames were taken every 10 s for 10 min. Video to Fig. 3 A.
• Video 10 -
  Trapping of collagen beads. Collagen I–coated red fluorescent polystyrene beads (red) were monitored by time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) at the apical surface of nonconfluent MDCK cells stably transfected with Lifeact-GFP (green). Bar, 2 µm. Frames were taken every 2 s for 200 s. Video to Fig. 8 C.
• Video 7 -
  Unbalanced dynamics of myosin. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) of a MDCK cell stably expressing MHCA-GFP, treated with 400 nM latrunculin B. Green, myosin at apical plane. Cyan, myosin at basal plane. Bar, 5 µm. Frames were taken every 10 s for 15 min starting 6 min after addition of latrunculin B. Video to Fig. 8 A.
• Video 6 -
  Disruption of the cell cortex. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) showing disruption of the apical MV array (left, cells stably transfected with Lifeact-GFP) and myosin network (right, cells stably transfected with MHCA-GFP) at the apical surface of MDCK cells, upon treatment with 2 µM latrunculin A. Maximum projections of three (for actin, Δz = 1.5 µm) or two (for myosin, Δz = 1 µm) focal planes are shown. Bar, 2 µm. Frames were taken every 20 s for 15 min. Video to Fig. 7 (A and B).
• Video 8 -
  Size-dependent confinement of beads. Carboxylated red fluorescent polystyrene beads (red) of indicated size were monitored by time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) at the apical surface of nonconfluent MDCK cells stably transfected with Lifeact-GFP (green). Bar, 2 µm. Frames were taken every 2 s for 200 s. Video to Fig. 8 (B and C).
• Video 2 -
  MV dynamics in nonconfluent cells. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) of the apical cortex region of a nonconfluent MDCK cell stably transfected with Lifeact-GFP to label actin. Bars, 2 µm. Frames were taken every 10 s for 10 min. Video to Fig. 3 B.
• Video 9 -
  Trapping of carboxylated beads. Carboxylated red fluorescent polystyrene beads (red) were monitored by time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) at the apical surface of nonconfluent MDCK cells stably transfected with Lifeact-GFP (green). Frames were taken every 2 s for 200 s. Video to Fig. 8 B.
• Video 3 -
  MV reorganization. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) showing examples of MV reorganization events in nonconfluent MDCK cells stably transfected with Lifeact-GFP: (1) bending, (2) exchange of connectivity, (3) flickering, and (4) lateral translation. Bar, 2 µm. Frames were taken every 10 s for indicated times. Red asterisks, arrowhead, and circles indicate ends of filaments described in the respective panels. Video to Fig. 3 (D–G).
• MATLAB souce codes (ZIP)
• Video 5 -
  Actin–myosin colocalization. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) of a nonconfluent MDCK cell double transfected with MHCA-GFP and Lifeact-mCherry. Bar, 2 µm. Frames were taken every 10 s for 15 min. Video to Fig. 6.
• Video 4 -
  Myosin dynamics. Time-lapse epifluorescence microscopy on a customized iMIC setup (FEI) showing isotropic reorganization of the apical myosin network in nonconfluent MDCK cells stably transfected with MHCA-GFP or MLC-GFP. Two focal planes (Δz = 1 µm) were maximum projected and color coded in red (most basal) followed by green and blue (most apical). Bars, 2 µm. Frames were taken every 10 s for 15 min. Video to Fig. 5 (E and F).