JCS042986 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. α-actinin and SF retraction. (a) Kymograph analysis of a SF retracting after laser dissection, showing the natural subunits movements and the edge detection (red curves). To resolve the 1 µm sarcomeric periodicity, we used a 150×/1,45 oil objective lens in combination with a 1.6× optovar zoom. (b) Time course of the subunits positions extracted from a) overlapped with the model curves, fitted with the parameter set (κ,δ,τ,τ_ε)=(0.082,0.89,7.5,0.21). (c) Normalized stripes width overlapped to the corresponding model curves calculated from b. (d) As previously described in Fig. 2 with the actin experiment, we compare the total SFs retraction ΔL (red data points, average with red segmented curve) to the calculated ΔL(κ, L). With α-actinin expressing cells, the ΔL distribution corresponds to a crosslink ratio κ < 0.078.

• Supplemental Figure S2 -

  Fig. S2. Correlative live cell imaging and TEM microscopy at focal adhesions. (a,d,h,i,j,l,n) All images correspond to the content of Figure 4. Additional information: (b) overview of the cell in TEM. Dashed rectangle show the corresponding two areas in a. (e-g) Closer view of a regular focal adhesion structure in TEM in three different planes. (k) Merge of planes P2 (from e, red) and P0 (from g, green). (m) The corresponding fluorescent micrograph with actin-Cherry in red and EGFP-zyxin in green.

• Supplemental Figure S3 -

  Fig. S3. Recruitment of vinculin at the sites of zyxin recruitment. (a-c) Sequences show a Ptk-2 cell, transfected with EGFP-zyxin and subject to SF nanosurgery. Zyxin sarcomeric localization vanishes quickly 1 second after cut in b to relocalize in random foci in c (the main ones are indicated with orange arrows). After fixation, correlative immunofluorescence with a Cy3 anti-vinculin antibody shows recruitment of vinculin at some of the sites, but not all as shown in e, where the EGFP expressed zyxin signal (conserved after fixation) is overlapped to the vinculin signal. Co-transfection of EGFP vinculin and Cherry-zyxin shows the same partial recruitment in j, 120 seconds after cut, as compared to the localization if vinculin prior to cut in g. The corresponding zyxin distribution after cutting several fibers is shown in i, merge of both labels in h and k. Scale bars: 5 µm (a), 10 µm (f).

• Supplemental Figure S4 -

  Fig. S4. Zyxin recruitment occurs from the cytosol. (a) Ptk-2 cell subject to sequential bleaching and laser dissection of EGFP zyxin labelled SFs. Bleaching of the full length of SFs, FAs included, performed inside the dashed red rectangle in b. Laser dissection occurs after the bleaching procedure along the dashed blue segment. (d,e) Recovery of FAs and recruitment along the SFs 5 and 100 seconds after cut respectively. (g-k) Closer views of a FA and part of an SF prior to bleach, (h-j) post-bleach and (i-l) 100 seconds post-dissection. (f) The intensity evolution of four foci along the SF and two connected FAs. FAs recovery was fitted with a single exponential function from the first point after bleach was completed, giving a recovery time reported in n (pink histogram). Zyxin foci intensity increase was fitted successfully with a single exponential from the first point after cut, yielding a threefold faster time constant in n (white bars). Control experiment of regular FRAP on FAs and foci intensity increase without bleaching on different cells is reported in m, showing a consistent threefold difference in dynamic recovery/increase time constants. Scale bar: 10 µm.

• Supplemental Figure S5 -

  Fig. S5. Discrete model for a SF: k_int and γ_int account for the internal visco-elastic properties of the SF, F_m denotes the contractile force of the motors at site n, and k_ext and γ_ext account for visco-elastic interactions with the surrounding cytoplasm. We consider a fiber fragment that is bound at x=0 and cut at x=L. The quantity a is the spacing of sarcomeric units of about 1 µm. The deduced model calculates the displacement µ along the SF.

• Supplemental Figure S6 -

  Fig. S6. The first row summarizes the results for fitting the full model with four parameters (κ=0.067, δ=0.58 µm, τ=52 seconds, τ_ε<0 seconds) to the kymograph data of Fig. 1e (main text). (A) Schematic of the model. (B) Model fit (solid lines) for the kymograph measured over 120 seconds. (C) Closer view of B with the first four stripes in the kymograph within 50 seconds after cut where most of the retraction happens. (D) Normalized stripe width. A simplified model excluding external friction within the cytosol (τ_εr0) produces the same fit results. (E-H) Equivalent analysis for the model, excluding internal friction (τr0, fit values: κ=0.041, δ=0.39 µm, τ_ε=0.71 seconds). Fitted curves (continuous lines) deviate from the experimental data. In contrast to the data, the inner bands retract slower than the bands closer to the cut (G,H). (I-K) Typical model curves neglecting cross-links (κr0, arbitrary values: δ=0.40 µm, τ=0.1 seconds, τ_ε=0.1 seconds). This model fails to reproduce the experimental data (fit to data not shown). In particular, each stripe is predicted to contract down to the same equilibrium length (K, for t>120 seconds), which was not observed in the experiment. The analysis shows that τ and κ are essential components of the model, but τ_ε plays a minor role.

• Movie 1 - Movie 1. Periodic photoleaching and nanosurgery show non homogeneous retraction of actin stress fibers.

• Movie 2 - Movie 2. Zyxin relocalization after SFs nanosurgery.

• Movie 3 - Movie 3. Zyxin relocalization after multiple SFs nanosurgery.

• Movie 4 - Movie 4. Dynamics of zyxin along after nanosurgery in the follicular tissue of a Drosophila egg chamber.

• Movie 5 - Movie 5. Zyxin translocates away from SFs after Myosin II inhibition with blebbistatin.

• Movie 6 - Movie 6. In situ SFs micromanipulation with AFM cantilever: α-actinin fluroescence intensity is stable upon mechanical load.

• Movie 7 - Movie 7. In situ SFs micromanipulation with AFM cantilever: zyxin fluorescence intensity reversibly increases upon mechanical load.