skTM depletes Arp2/3 and ADF/cofilin from the leading edge of PtK₁ cells. (A) Immunolocalization of Arp3 and F-actin (fluorescent phalloidin) in control cells (A) and cells injected with skTM (B). Lines in A are examples of one region where fluorescence intensity line scans were taken to produce the type of data seen in E and F. ADF/cofilin and F-actin localization in control cells (C) and cells injected with skTM (D). Boxed regions are magnified in the Merge column, with Arp3 (A and B) and ADF/cofilin (C and D) in green and F-actin in red. (E) Average fluorescence intensity ratio of Arp3/F-actin and cofilin/F-actin in control and skTM-injected cells measured from the cell edge (0 μm) into the cell center. Fluorescence intensity of Arp3 (F) or ADF/cofilin (G) and F-actin staining in control and skTM-injected cells measured from the cell edge (0 μm) into the cell center. In E–G, data are averaged from 13 cells per treatment, three measurements per cell.

skTM inhibits the formation of the lamellipodium. (A) F-actin turnover maps computed from qFSM time-lapse movies and corresponding phase-contrast images in a control, an skTM-injected (skTM), and a GFP-CA-expressing (GFP-CA) cell. Turnover maps depict polymerization (red) and depolymerization (green) rates. Control cells exhibit narrow bands of fast polymerization and depolymerization in the lamellipodium adjacent to the leading edge (arrow) and spatially random punctae of polymerization and depolymerization in the lamella. Cells containing skTM or GFP-CA exhibit lamella F-actin kinetics at their edges (arrows). (B) FSM image of F-actin in a control PtK₁. White line shows location used to generate kymograph in C. (C and D) Kymographs of a control (C) and an skTM-injected (D) cell; white lines highlight speckle translocation used to calculate flow velocities. There are two regions of distinct retrograde flow rates in controls and only one region of retrograde flow in cells containing skTM. (E and F) Kymographs taken from F-actin FSM time series of skTM-containing cells. In E, at the first arrowhead, 200 nM cytochalasin D was perfused and washed out at the second arrowhead. Arrows illustrate leading edge protrusive behavior; lines emphasize retrograde flow rates. In cytochalasin D, leading edge protrusion stops, but retrograde F-actin flow continues. In F, 100 μM blebbistatin was perfused at the arrowhead; lines emphasize actin flow rates. After treatment, retrograde and anterograde F-actin flow immediately arrested. (G) Average F-actin flow rates in different cellular regions taken from kymographs of control (blue) and skTM-injected cells (red) (bars equal SEM). Negative and positive flow rates imply retrograde and anterograde flow, respectively. Convergence rate is the sum of the absolute value of average lamella retrograde flow and cell body anterograde flow.

skTM increases leading edge protrusion persistence and cell migration velocity. Phase-contrast images (left column) and kymographs (right two columns) taken from movies of leading edges of control cells (top row) and skTM-injected cells (bottom row). White lines depict locations used to generate kymographs. Arrowheads delineate the beginning of each retraction phase. Graph displays average instantaneous cell migration velocity in control, skTM-injected (skTM), and GFP-CA (CA)–expressing PtK₁ cell islands. *, P < 0.01.

skTM enhances the stiffness of the lamella. Phase-contrast micrographs of a control cell (A) and an skTM-injected cell (B) overlaid marks corresponding to the positions of fluorescent microspheres that were injected into the cell. Particle loci are color coded according to the local value of the cytoplasmic elasticity from red (softest) to blue (stiffest). (C) Relative elasticity of the cell body (CB) and lamella (LM) of control and TM-injected cells calculated at a sampling frequency of 1 Hz. Graph shows mean elasticity (± SEM); differences in elasticity are significant (P < 0.05).

skTM localizes to F-actin throughout the cell. (A) Island of PtK₁ epithelial cells. (B) Western blot analysis of PtK₁ lysate for TM isoforms. (C) Purified skTM seen by Coomassie blue. Numbers refer to mass markers. (D) Immunolocalization of long TMs (TM311 monoclonal) and F-actin (fluorescent phalloidin) in a PtK₁ cell. TM is excluded from the cell edge. (E) PtK₁ cell injected with skTM and immunolabeled with muscle-specific CH1 antibody and costained for F-actin. Filopodial-like protrusions occur in cells injected with skTM (arrowhead). Merged images in D and E are from boxed regions, with F-actin and TM in red and green, respectively. Whole mount EM of control (F and G) and skTM-injected (H and I) cells. G and I show enlargements of F and H, respectively. Near the cell edge, an isotropic network and small vertical ruffles (arrows) are present, followed by the lamella with dense population of bundles (yellow) oriented toward the cell margin. A horizontal dense belt of filaments (red) delineate the cell body from the lamella (red). In the injected cell (H), the only visible network is comprised of oriented bundles (yellow) and filopodia-type protrusions (arrow in I). Bars: (F and H) 2.4 μm; (G and I) 5.0 μm.

skTM decreases the concentration of polymerization-competent free barbed filament ends at the cell edge. Barbed-end actin incorporation (green) and fluorescent phalloidin (red) in a control cell (A) and an skTM-injected cell (B). (C) Intensity ratio of fluorescent actin incorporation marking free barbed filament ends relative to F-actin. (D) Intensity of fluorescent actin incorporation marking free barbed ends and F-actin staining. Profiles in C and D were measured from line scans taken from the cell edge (0 μm) into the cell center and data are averaged from 14 cells per treatment, three measurements per cell.

skTM recruits myosin IIA to the leading edge. Immunolocalization of myosin IIA heavy chain and fluorescent phalloidin staining of F-actin in a control (A) and an skTM-injected (B) PtK₁ cell. The insets are magnified in the right column, with F-actin red and myosin IIA green. (C) Intensity ratio of myosin IIA/F-actin in control and skTM-injected cells measured from the cell edge (0 μm) into the cell center. (D) Fluorescence intensity of myosin IIA and F-actin staining in control and skTM-injected cells measured from the cell edge (0 μm) into the cell center. In C and D, data are averaged from 14 cells, three measurements per cell.

Substrate adhesions form closer to the leading edge in cells containing skTM. F-actin staining (fluorescent phalloidin, red) and paxillin immunolocalization (green) in control (A) and skTM-injected (B) cells. Arrowhead in B shows paxillin punctae along central F-actin bundles that are not present in F-actin in A. (C) Frequency histogram of the distance of the distal-most border of paxillin foci from the leading edge in control and skTM-injected cells (n = 8 cells, 10–15 measurements per cell per treatment). Numbers in the top right of graph are the mean distance (± SEM) of the adhesion sites from the cell edge.

Antibody inhibition of long TM isoforms displaces TM from F-actin and decreases myosin II–dependent F-actin convergence. (A and B) F-actin (fluorescent phalloidin) and long isoforms of TM (TM 311 antibody) in control (A) and cells injected with TM311 antibody (B). In control cells, long TM localizes along F-actin bundles, but is displaced from F-actin bundles in TM311 antibody–injected cells. Right column shows a magnified view of TM in the boxed regions. (C) Average F-actin flow rates ± SEM in control and TM311-injected cells with negative and positive rates indicating retrograde and anterograde flow, respectively.