Vimentin fibers orient traction stress

Abstract

The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or "fibers," to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.

Keywords: fiber orientation; intermediate filaments; mesenchymal migration; traction stress; vimentin.

Fig. S1.

TALEN-edited mEmerald-vimentin expression and cell migration in primary hFFs. (A, Top) Western blot quantification of vimentin protein expression in WT and TALEN- modified cells. Cell lysates from WT and genome-edited hFFs were twofold serially diluted, and six samples from the dilution series for WT and edited lysates were run and blotted on the same membrane. Membranes were probed with anti-vimentin together with loading control antibodies for GAPDH for normalization. Blots were scanned on an infrared imaging system, and band intensities were quantified with Image Studio Light 4.0 software (LI-COR). Scans for mouse anti-Vimentin (clone V9; Sigma-Aldrich) and rabbit anti-GAPDH (rabbit GTX100118; GeneTex) are shown. The expected molecular weight of vimentin is 54 kDa, that of mEmerald-vimentin is 70 kDa, and that of GAPDH is 37 kDa. Intensities for bands corresponding to untagged and fusion proteins and the sum of both are plotted. The band intensities suggest that vimentin protein levels are roughly equal in parental and edited cells, and that approximately 10% of vimentin is tagged with fluorescent protein mEmerald. (A, Bottom) Results were confirmed with a second rabbit antibody against the C-terminal peptide of vimentin (C20; Santa Cruz Biotechnology). The GAPDH (mouse GT239; GeneTex) loading control from the same blot is shown. Compared with the parental cell line, in the edited cells, mean vimentin expression was 105 ± 54% (n = 9 blots, two different antibodies). In the edited hFF cell line, 4 ± 4% vimentin was tagged with mEmerald. This could indicate that the fusion protein was repressed or less stable and subject to proteolytic degradation. The high SDs reflect the variation between blots and between the different antibodies. (B, Left) TALEN-edited cells migrated at the same speed as control lentiviral cells, whereas lentiviral knockdown significantly decreased migration speed (P < 0.01). n = 100 cells for each variable. Cells were plated on glass-bottom MatTek dishes coated with 10 μg/mL fibronectin and imaged concurrently for 8 h in an ambient-controlled chamber. Values are mean from four experiments. (B, Right) Western blot of vimentin (clone V9; Sigma-Aldrich) and rabbit anti-GAPDH (GTX100118; GeneTex) from control and vimentin kd lentivirus-transfected cells. The average knockdown efficiency in two separate experiments was 50%.

Fig. S2.

WT hFFs (A) and TALEN genome-edited hFFs (B) appear similar in morphology and vimentin distribution. Cells were plated on glass coated with fibronectin and allowed to spread for 4 h before fixation and staining with anti-vimentin antibody. (Scale bar: 10 μm.)

Fig. 1.

Characterization of the vimentin network by computational image processing. (A) Z-stack maximum intensity projection of mEmerald-vimentin distribution within an hFF cell. (B) Vimentin mesh (Left and Middle images) and fibrous architecture (Middle and Right images). All images are z-stack maximum intensity projections of mEmerald-vimentin in three representative cells. (Insets) 2× zoom of the areas of detail. (C) Raw spinning disk confocal image of mEmerald-vimentin in an hFF cell. (D) Multiscale steerable filtering was applied to the image in C to enhance curvilinear features. (E–G) Extraction of vimentin fibers. (E) Nonmaximum suppression was applied to D, followed by iterative graph matching of skeletonized curvilinear features in close proximity and orientation to extract a map of network fragments of various lengths. (F) Fragments from E meeting an experimentally determined length threshold (4 μm) were classified as vimentin fibers (magenta). All high- confidence pixels from D not classified as fibers were classified as mesh (gray). (G) Inset of F showing the raw image (Top) and analyzed pixel map (Bottom) of fibrous and mesh-like vimentin. (H) Orientation of vimentin fibers in the image frame of reference. (Scale bar: 10 μm.)

Fig. S3.

Vimentin network organization subject to actin and MT depolymerization. (A–C) Cells were subjected to the following treatments at 5 min prior to fixation and staining: DMSO control (A), 1 μM cytochalasin D (B), and 500 nM combretastatin (C). (Top) Raw mEmerald-vimentin in treated hFFs. (Middle) Pixel maps of fibrous vimentin (magenta) overlaid on mesh (gray). (Bottom) Raw MT (Left) and raw actin (Right) images. (Scale bars: 10 μm.) (D and E) Analysis of network architecture for a given treatment. DMSO, n = 17 cells; cytochalasin D, n = 26 cells; combretastatin, n = 9 cells. (D) Percentage of an individual cell covered by fibrous vs. mesh vimentin. (E) Mean straightness of all detected fibers within a single image. Error bars indicate SD.

Fig. S4.

The fiber/mesh ratio in migrating cells is stable over time. The fiber/mesh pixel ratio was measured over the time course of an entire movie for each cell, giving an estimate of fiber structure stability. (A) The mean fiber ratio for all cells (n = 53) was 0.21 (solid line), with an SD of 0.11 (dotted lines), demonstrating variability within the population. (B) The fiber/mesh ratio was stable within individual cells. Shown is the ratio from a representative migrating cell. (C) The percent change in fiber ratio within a cell was calculated as SD/mean. The median change in fiber ratio within cells was 7.5% (solid line).

Fig. 2.

Vimentin network geometry during mesenchymal migration. (A) Vimentin fiber orientation in migrating hFFs. The arrow points to the same cell over time. Note that the image contrast of this time lapse was adjusted to more clearly visualize peripheral vimentin fibers (Methods). (B) hFFs expressing mEmerald-vimentin were stained with a 642-nm emitting membrane dye to measure cell shape and position. The outline of cells was masked and then skeletonized (Methods). The cell position was determined by the center of the cell mask. The orientation of cell movement was tracked from frame to frame. (C, Left) The angles between the branches of the skeletonized cell shape and vimentin fibers in the same region of the cell were compared for each branch in each frame. Shown are all individual measurements over all time-lapse sequences (8-h time lapse; n = 60 cells). Data are mean values from four separate experiments. The measured distribution deviates from a uniform distribution with P < 10⁻⁵ (Kolmogorov–Smirnov test). (C, Right) The mean vimentin fiber orientation angle within a cell was also compared with the orientation of whole cell movement. The measured distribution deviates from a uniform distribution with P < 10⁻⁵ (Kolmogorov–Smirnov test). (Scale bar: 10 μm.)

Fig. 3.

Vimentin network relative to actin flow speed. TALEN-modified hFFs expressing mEmerald-vimentin and transiently expressing actin SNAP-tag were incubated with SNAP-TMR. Both wavelengths were imaged every 5 s. Actin behavior was analyzed using qFSM software (22). (A) SNAP-TMR actin distribution in hFFs. Zoom-in of inset is the raw vimentin image overlaid with the cell outline. (Scale bars: 5 μm.) (B) Detected vimentin fibers overlaid with actin flow vectors (white). (C) Zoom-in of boxed regions in B. Vimentin fibers are shown in red. Each white pixel represents the position of an identified actin speckle. Vector length represents the time interval over which a speckle was tracked. (D) Actin flow speed vs. colocalized vimentin polymer organization (none, mesh, fiber) for n = 7 cells. Shown from left to right: no vimentin, M = 5,046 actin flow tracks; mesh, M = 14,130 flow tracks; and filamentous vimentin, M = 418 flow tracks.

Fig. S5.

Effects of the vimentin network on actin flow speed and orientation. TALEN-modified hFFs expressing mEmerald-vimentin and actin SNAP-tag were incubated with SNAP-TMR to label actin. Both wavelengths were imaged every 5 s. Actin behavior was analyzed using QFSM software as described previously (22). (A) Actin flow speed vs. vimentin intensity throughout the vimentin network. Actin flow data are separated by distance from the center of the vimentin network (left to right, in μm) and by vimentin architecture (top to bottom). (B) The orientation of actin flow vectors was compared with that of colocalized vimentin fibers for a single cell (Left) and for seven cells (Right). The Kolmogorov–Smirnov test indicated no evidence of co-orientation between actin flow vectors and vimentin fibers.

Fig. 4.

Vimentin network effects on traction forces. (A, Left) Pseudocolor image of traction force exerted by an hFF expressing control lentivirus. (A, Right) Traction force exerted by an hFF expressing shRNA targeting vimentin. Traction force range (minimum to maximum) was adjusted to display the distribution effectively. In the left panel, the range corresponds to 0–0.8 kPa; in the right panel, the range corresponds to 0–1.0 kPa. (Scale bar: 5 μm.) (C, Left) Mean number per cell of traction force peaks in the interior (area >7 μm from the cell edge; Methods) of control and vimentin kd cells. **P = 0.0115, t test. (C, Right) Mean number of traction peaks in the 7-μm-wide peripheral band. Control cells, n = 20; kd cells, n = 19. Error bars represent SEM. (D and E) Zoom-in views of boxes indicated in A, showing vimentin distribution (Left) and traction force distribution (Right). (D) I; (E) II. (F) Interior (Top) and peripheral (Bottom) traction stress vs. colocalized vimentin intensity for representative control (Left) and kd (Right) cells. The counts of traction stresses for given magnitude and vimentin intensity are presented on a pseudocolor scale, with a range of 0–3000.

Fig. 5.

Effect of vimentin fibers on traction stress orientation. (A, Top) Segmented vimentin fibers color-coded for orientation and traction stress vector orientation for control cell shown in Fig. 4A. (A, Bottom) Zoom-in of box indicated in the top panel. (Scale bars: 5 μm.) (B and C) Illustration (B) and measurement of (C) angular differences between vimentin fibers and colocalized traction force vectors for n = 12 cells. The measured distribution deviates from a uniform distribution with P < 10⁻⁵ (Kolmogorov–Smirnov test). (D) Local alignment of traction stress vectors within a circular window of 2.5-μm radius measured in control and vimentin kd cells in regions with and without detectible vimentin. Control cells, n = 12; kd cells, n = 9. (E) Model of the vimentin network as a load-bearing superstructure that redirects actomyosin forces to the peripheral adhesions and aligns the traction force field with the orientation of the vimentin fibers.