Research Papers

The Mechanical Contribution of Vimentin to Cellular Stress Generation

[+] Author and Article Information
Inge A. E. W. van Loosdregt

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: i.a.e.w.v.loosdregt@tue.nl

Giulia Weissenberger

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven MB 5600, The Netherlands
e-mail: g.weissenberger@student.tue.nl

Marc P. F. H. L. van Maris

Department of Mechanical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: m.v.maris@tue.nl

Cees W. J. Oomens

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: c.w.j.oomens@tue.nl

Sandra Loerakker

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: s.loerakker@tue.nl

Oscar M. J. A. Stassen

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: o.m.j.a.stassen@tue.nl

Carlijn V. C. Bouten

Department of Biomedical Engineering,
Eindhoven University of Technology,
P.O. Box 513,
Eindhoven 5600 MB, The Netherlands
e-mail: c.v.c.bouten@tue.nl

Manuscript received September 19, 2017; final manuscript received January 26, 2018; published online March 21, 2018. Assoc. Editor: Nathan Sniadecki.

J Biomech Eng 140(6), 061006 (Mar 21, 2018) (10 pages) Paper No: BIO-17-1418; doi: 10.1115/1.4039308 History: Received September 19, 2017; Revised January 26, 2018

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

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Grahic Jump Location
Fig. 1

Overview of the experimental design of this study, including the different techniques that were used

Grahic Jump Location
Fig. 2

Representative fluorescence microscopy images of vimentin (green; (a)–(d)), α-tubulin (red; (e)–(h)), and nuclei (blue) of MEFs cultured on perpendicular and parallel substrates (for clarity fibronectin is not shown). Scale bar is 20 μm. (Color version online).

Grahic Jump Location
Fig. 3

Representative fluorescence microscopy images of vinculin (red) and nuclei (blue) of MEFs cultured on perpendicular ((a), (b)), parallel ((c), (d)), and homogeneous substrates ((e), (f)); fibronectin in gray. Scale bar is 20 μm. Quantification of the focal adhesion size (mean ± standard error of mean) showed no significant differences between the focal adhesions of the four different experimental groups, while the focal adhesions of the homogeneous substrates were smaller compared to the patterned substrates (g). ***: significantly lower than all patterned substrates with p < 0.001, #: significantly lower than VimWT MEFs on perpendicular substrates and VimKO MEFs on both patterned substrates with p < 0.05 (n = 3–4 fluorescent microscopy images, leading to >1300 focal adhesions per group). (Color version online).

Grahic Jump Location
Fig. 4

Representative fluorescence microscopy images ((a)–(d)) of actin (green), and nuclei (blue) of MEFs cultured on perpendicular and parallel substrates (fibronectin in gray) and the corresponding orientation histograms ((e)–(h); mean ± standard error of mean) of nuclei (blue markers) and actin (green markers). Scale bar is 50 μm, n = 40 fluorescent microscopy images per group. Corresponding order parameter ((i) and (j); mean ± standard deviation) of the nuclei ((i); blue), and actin fibers ((k); green). *: significantly different from VimWT parallel, and #: significantly different from VimKO parallel. Triple symbols represent p < 0.001. (Color version online).

Grahic Jump Location
Fig. 5

Examples of thin films seeded with wild-type MEFs on parallel substrates that fall within ((a); both films, (b); right film) and above ((b); left film) the measurement limit. Graphical representations of the projection length (white bars) and the original length (black bars) are included in the images. Scale bar is 1 mm. Top view (c) and side view (d) images of examples of deformed finite element meshes at increasing levels of σnet (from left to right). The red elements represent the cell layer; the blue elements represent the PDMS layer. The undeformed mesh is shown in gray. (Color version online).

Grahic Jump Location
Fig. 6

(a) Percentage of thin films that fall within and above measurement limits. Of the films within measurement limits, σnet was determined at 0 h (b) and 1 h (c), this is depicted against the cell density. The cell density of the films that fall above the measurement limit is also depicted in (b) and (c). Total number of films created is 34 for VimWT perpendicular, 45 for VimWT parallel, 41 for VimKO perpendicular, and 48 for VimKO parallel.

Grahic Jump Location
Fig. 7

σnorm (=σnet/cell density) is significantly higher in VimKO MEFs compared to VimWT MEFs. Symbols indicate significant differences, with single symbol indicating p < 0.05, and double symbols indicating p < 0.01. *: significantly higher than VimWT perpendicular at the same time point, #: significantly higher than VimWT parallel at the same time point. n = 23–43 at 0 h and 15–39 at 1 h.

Grahic Jump Location
Fig. 8

Representative immunofluorescence microscopy images of actin (green), pMyosin (red), and nuclei (blue) of MEFs cultured on perpendicular and parallel substrates (fibronectin in gray). The top row (a)–(d) shows the merged images, the second row (e) and (f) shows actin and nuclei, the third row (i)–(l) shows pMyosin and nuclei, and the last row (m)–(p) shows higher magnification images of the boxes indicated in (e)–(l). Scale bar is 20 μm. (Color version online).




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