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First published online 28 April 2009
doi: 10.1242/jcs.042986

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Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization

Julien Colombelli^1,*,, Achim Besser², Holger Kress^1,3, Emmanuel G. Reynaud¹, Philippe Girard¹, Emmanuel Caussinus⁴, Uta Haselmann¹, John V. Small⁵, Ulrich S. Schwarz² and Ernst H. K. Stelzer¹

¹ Cell Biology and Biophysics, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany
² University of Heidelberg, Bioquant, BQ0013 BIOMS Schwarz, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany
³ Department of Mechanical Engineering, Yale University, New Haven, CT 06511, USA
⁴ Biozentrum, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland
⁵ Institute of Molecular Biotechnology Austrian Academy of Sciences (IMBA), Dr Bohrgasse 7, A-1030, Vienna, Austria

View larger version (87K): [in this window] [in a new window]   Fig. 1. Actin SF retraction after photobleaching and laser nanosurgery. (A) A 3 µm periodic laser pattern (dashed lines) bleaches across a Ptk-2 cell expressing actin-EGFP, perpendicularly to the SF axes. Laser dissection (blue lines) occurs between two stripes within 10 to 20 seconds after bleaching. (B) 120 seconds after cut. (C) SFs expressing actin-Cherry and -actinin-EGFP. (D) 120 seconds after laser release. Note the stronger retraction at the tips. (E,G) Kymograph analyses of SFs from both experiments. The edge detection performed for further analysis of the stripes displacement is shown in red. L, total retraction of the fiber after equilibration. (F,H) Normalized widths of five subunits closer to the cut tips, numbered according to kymographs. (I) L as a function of the initial SF length L, measured in epithelial cells and fibroblasts. Segmented lines show mean values calculated over 4 µm intervals of the x-axis, bars show s.d. L reaches a constant value for L>10 µm. (J-N) SF dissected simultaneously in two locations. The retraction occurs from both locations and is shown 2 seconds, 10 seconds and 60 seconds after cut in K, L and M, respectively. (N) Overlap of the three time points with red (K), green (L) and blue (M) shows no lateral movement of the fiber fragments as they retract along their original axis. Scale bars: 10 µm (B), 5 µm (D), 3 µm (E), 1 µm (G). See supplementary material Movie 1.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Viscoelastic model used for data analysis. (A) Schematic view of the internal SF organization with actin, myosin II and the actin crosslinker -actinin. (B) Representation of the viscoelastic model. In red, the elements of the internal contractile units: spring of stiffness kint, dashpot of viscosity int and contractile force Fm. In green, the external anchor springs of stiffness kext. Cytosolic friction is denoted by ext. FAs and anchoring substrate are considered hard boundaries. Each kymograph was fitted with one set of model parameters. (C) Time course of the bleached stripes positions extracted from Fig. 1E overlapped with the fitted model curves. (D) Normalized stripe width overlapped to the corresponding model curves calculated from C. Colors correspond in C and D. One set of the four model parameters is sufficient to reproduce the non-homogeneous retraction of the whole SF. Fit values for parameters in C and D are (, , , ) = (0.067, 0.58 µm, 52 seconds, 0.0 seconds). (E) In red, the averaged total retraction L(L), reported for Ptk-2 cells in Fig. 1I, shows a very good agreement with the model curve (green), averaged with 86 parameter sets extracted from the actin data (error bars show s.d.). The L(L) model curve corresponds to an average crosslink ratio 0.034 (see Appendix 1). For comparison, other L curves are plotted (dashed lines) with =1, 0.005, 0.0001 and 0. Note that =0 would correspond to a free SF, i.e. with no external linkage (kext=0).

View larger version (88K): [in this window] [in a new window]   Fig. 3. Force correlates with protein localization along SFs. A Ptk-2 cell transfected with Cherry-zyxin and actin-EGFP constructs, before (A) and after (B,C) SF dissection (blue lines). (B) A 14 µm SF fragment is first released and retracts. (C) A second cut dissects the fragment, in its middle, and a neighbor control SF. (D) zyxin before cut, (E) zyxin 6 seconds after cut, (F,G) actin and zyxin 30 seconds after cut, (H,I) 30 seconds after second cut. Blue arrows indicate laser positions; red arrows indicate direction of retraction. The typical striated zyxin pattern (D) is lost instantaneously after release (E). (J) Intensity along the cut SF shows the loss of periodic signal along the SF axis in the 10-25 µm region. (K) Zyxin intensity in selected regions of C, numbered 1,2,3,4 for sites along SFs, and FA1 and FA2 for selected FA. Foci 1 and 2 (red) connected to the released fragment show fast enrichment of zyxin, which reverses after the second cut concomitantly with the reverse of actin movement (red arrows, see also M and supplementary material Movies 2-3). Foci 3 and 4 connected to outer retracting fibers undergo constant increase (green). FA2 (blue) shows important loss of zyxin, also visible in J, whereas the control FA is stable. (L) Zyxin intensity loss (right axis) at FAs connected to cut SFs, quantified in Ptk-2 cells (red, n=74 data points, segmented line: average ± s.d.) and 3T3 fibroblasts (blue, n=88, only average ± s.d.). The curves are in good agreement with the calculated force loss F(,L) (Appendix 1) obtained from the SF analysis in Fig. 2, which yielded 86 datasets and values of . The green curve shows the force loss average (± s.d.). (M) Double color kymograph of the central fiber fragment (red: actin, green: zyxin). (N) Intensity profile of the SF fragment, normalized and color coded according to the color scale (left of N). (O) Normalized calculated tension (x,t)/0 within the SF fragment. (P) Traction Ftrac(x,t) normalized by maximal occurring value after cut (Appendix 1). The same normalized color scale applies to O and P. Symbols indicate common features in the experiments and the model. In O, tension is lost after cut (symbols + and –); in P, traction is built onto substrate crosslinkers (*) in a reversible manner after the second cut as shown by the subsequent loss (x). (O,P) contribute together to the observed zyxin intensity along the SF (N). Parameter values used in O and P are (, , , ) = (0.01, 0.70 µm, 5 seconds, 0.1 seconds). Kymographs M,N are interpolated. Scale bars: 5 µm (A); 5 µm horizontally, 10 minutes vertically (O). In both FAs and SFs, there is a general dynamic correlation between force and zyxin localization.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Correlative TEM and fluorescent live microscopy shows that zyxin localizes at the basal membrane after cut. A Ptk-2 cell cotransfected with EGFP-zyxin and actin-Cherry constructs, 20 seconds after laser nanosurgery. (B) Schematic view of the Epon sectioning orientation. Plane P0 includes the basal side at the glass surface (0-50 nm), P1 the second (50-100) and P2 the third (100-150 nm). TEM views (inverted contrast) of the region in A (dashed rectangle): P2 in C, P1 in D, P0 in E. (F) Merged planes P0(green)+P1(red). (G) Merged planes P0(green)+P2(red). (H) Comparison of F and G to view fluorescent zyxin (green) and actin (red) in shows that zyxin localizes in the first section at the basal membrane. (Note that dot-like structures appearing sparsely in P0 are likely to be dirt particles on the Epon block surface.) Scale bars: 5 µm (A), 1.5 µm (E), 1 µm (H). See also supplementary material Fig. S2.

View larger version (99K): [in this window] [in a new window]   Fig. 5. The dynamics of zyxin along actin bundles in follicle cells of a Drosophila egg chamber. (A) Schematic view of a stage 11 egg chamber. (B) Polygonal organization of cells at apical side, axial projection of confocal stack. (C) Actin organization at the basal side. Actin-rich parallel bundles resemble SFs. (D,E) split channels with expressed Ypet-zyxin and red phalloidin staining. (F) Widefield fluorescent image of a live egg with Ypet-zyxin showing zyxin-rich areas along the cell outline. (G,H) Comparison of the cell intensity signal before and after laser surgery (blue target). Intensity is color-coded with the lookup table `Red Hot' in ImageJ. A signal increase close to the cut is clearly shown in H, along the position of the actin bundles (white arrowheads). (I) Intensity dynamics of several areas, noted in F as areas 1,2,3 and 4. Areas belonging to the cut cell undergo a detectable decrease of fluorescence whereas control cells undergo random and weak fluctuations. See also supplementary material Movie 6. Scale bars: 10 µm (B,E).

View larger version (85K): [in this window] [in a new window]   Fig. 6. Myosin-dependent localization of zyxin in SFs. Myosin II inhibitor blebbistatin (10 µM) was applied to Ptk-2 cells cotransfected with EGFP-zyxin and actin-Cherry (A-H) and with -actinin-EGFP and Cherry-zyxin (I-P). (A,I) Overviews of the cells prior to treatment, (B,J) 10 minutes after treatment. (C-H,K-P) Closer view of the boxed areas. The initial sarcomeric localization of zyxin (E,M) vanishes (F,N) whereas actin SFs (C) and -actinin (K) are stable 10 minutes after treatment (D,L). (Q-U) Control with DMSO only, showing no change in localization of zyxin and -actinin. Scale bars: 10 µm (A-I), 5 µm (H-P). See also supplementary material Movie 7.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Acute recruitment of zyxin on single SFs pulled with an AFM cantilever. (A) Position of the fibronectin-coated AFM cantilever, extracted from a transmission micrograph taken before traction, overlapped with a EGFP-zyxin-expressing Ptk-2 cell. In inset, schematic side view of the manipulation experiment with the AFM tip in orange, applied from the top and connecting to basal SFs through the membrane. Springs represent SF subunits. Traction applies to the neighboring cell's SFs and propagates to fluorescent SFs of interest through cell-cell junctions (white arrows). Two fibers are periodically pulled over a distance x (See supplementary material Movie 7). (B) Relaxed and pulled fibers during two different cycles. (C) Kymograph showing the full traction sequence over nine cycles. (D) Zyxin intensity increase over time (left, red) compared with a control fiber (green). x was manually measured from the kymograph. The total cell intensity was corrected for bleaching (see background in blue). The intensity of pulled and control SFs in -actinin-expressing cells (right, see supplementary material Movie 6), showing no correlation between the traction (top) and the intensity of the SFs. (E) Intensity profile of the seventh traction plotted before traction (dashed yellow, reported with offset in plain yellow because of overlap) and during the traction (red). Before traction, the typical sarcomeric labeling of zyxin is clearly visible from the intensity profile with, on average, 1 µm periodic ripples. After traction, the amplitude of the ripples increases in the front stretched part of the fiber, thereby showing a discrete and periodic recruitment of zyxin along the bundle. Scale bars: 5 µm (A-C) horizontal, 40 seconds vertical (C).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Possible mechanism of mechanosensing in SFs. Zyxin is recruited along actin bundles and FAs by mechanical force increase. In SFs, the opposite contraction force (red arrows) applied by myosin II induces the opposite movement of antiparallel actin filaments, which could be transduced to the actin-actin crosslinker -actinin. A conformational change or binding properties modifications could allow zyxin recruitment. At FAs (bottom left), the same contraction movement can propagate to -actinin, which links actin filaments and FA proteins such as vinculin. The mechanical switch regulating zyxin recruitment could have the same force-transduction mechanism with the same molecule, for example -actinin, in SFs and FAs.

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