JCS008219 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. COS7 cells expressing EGFP-supervillin, but not those expressing EGFP alone, spread slower than untransfected cells. Micrographs of COS7 cells transfected with EGFP-SV (left panels) or EGFP (right panels) after plating onto 10 μg/ml fibronectin for 15, 30, 45, 60 or 90 minutes, as indicated. EGFP signals (left panels) and phase contrast images (right panels). Scale bar, 50 μm.

• Supplemental Figure S2 -

  Fig. S2. Blebbistatin, ML-7 and U0126, but not Y-27632, antagonize supervillin-associated bundling of myosin II and F-actin at the peripheries of COS7 cells. Micrographs showing EGFP-SV (a,d,g,j,m), myosin IIB (b,e,h,k,n) and F-actin (c,f,i,l,o) localizations during cell spreading in the absence (a-c) and in the presence of drugs that specifically inhibit myosin ATPase activity (d-f; 20 μM blebbistatin); MLCK (g-i; 1 μM ML-7); the MLCK activator, MEK (j-l; 2.5 μM U0126); or Rho kinase (m-o; 25 μM Y-27632). Myosin IIB was visualized in the red channel, and F-actin with Alexa-Fluor-350-phalloidin. Bar, 20 μm.

• Supplemental Figure S3 -

  Fig. S3. Low concentrations of ML-7 or U0126 inhibit the supervillin-mediated recruitment and/or phosphorylation of MLC at the peripheries of COS7 cells. Micrographs showing EGFP-SV (a,d,g,j,m) in the green channel and pSer19-MLC (b) or pThr18pSer19-MLC (e,h,k,n) in the red channel. In the merged images (c,f,i,l,o), signal overlaps appear as yellow or orange. Bar, 20 μm.

• Supplemental Figure S4 -

  Fig. S4. ML-7 at high concentrations destroys myosin II, F-actin and focal adhesion structure during cell spreading. Micrographs showing A549 cells treated with 10 μM ML-7 during spreading, as described in Figs 3 and 7, fixed and stained with antibodies specific for MHC IIA (a), vinculin (c, f), MHC IIB (d), mono-phosphorylated Ser19-MLC (pS19-MLC, g) or di-phosphorylated Thr18Ser19-MLC (pT18pS19-MLC, h). F-actin was visualized with Alexa-Fluor-350-conjugated phalloidin (b,e,i). Bar, 10 μm.

• Movie 1 -

  Movie 1. Time-lapse (1 minute interval, 20 minutes) phase-contrast movie of an A549 cell treated with control dsRNA during the linear phase of spreading on fibronectin. The rate of spreading, which is near the average for control cells, was 36.2 μm²/min. Micrograph, 50 μm × 50 μm.

• Movie 2 -

  Movie 2. Time-lapse (1 minute interval, 20 minutes) phase-contrast movie of an A549 cell treated with hSV2 dsRNA during the linear phase of spreading on fibronectin. The rate of spreading, which is near the average for hSV2-treated cells, was 47.6 μm²/min. Micrograph, 50 μm × 50 μm.

• Movie 3 -

  Movie 3. Time-lapse movie of EGFP-tagged SV1-171 in transfected COS7 cells. Notice the initial association with linear stress fibers and the breakage of these structures into elongated punctae during cytoskeletal remodeling. Bar, 10 μm; elapsed time: 40 minutes.