1. Mechanotransmission and Mechanosensing of Human alpha-Actinin 1
Shimin Le, Xian Hu, Mingxi Yao, Hu Chen, Miao Yu, Xiaochun Xu, Naotaka Nakazawa, Felix M. Margadant, Michael P. Sheetz, Jie Yan 
Cell Reports 
Volume 21, Issue 10, Pages (December 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2714-2723DOI: (10.1016/j.celrep.2017.11.040)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Force-Responses of Full-Length Human α-Actinin 1 and the CaM Domain
(A) Left: the structure of α-actinin 1, showing the functional domains: ABD, the rod domain, and the CaM like domain with PDB structures (Atkinson et al., 2001; Liu et al., 2004) based on smooth muscle isoform. Right: illustrative sketches of the experimental design and five constructs containing the full-length α-actinin 1, the EF-hands, the rod domain, double-rod domain, the SR2-3 domain, respectively.
(B) Typical force-extension curves of full-length α-actinin 1 unfolding at a loading rate of ∼1 pN s−1 and the subsequent refolding at a loading rate of ∼−0.3 pN s−1. The eight red arrows indicate the domain unfolding events during force-increase scan. The inset shows the refolding events during the force-decrease scan.
(C) Left: typical force-extension curves of the CaM domain (containing EF12 and EF34) at a loading rate of ∼0.5 pN s−1. Right: the transition (unfolding/refolding) force and stepsize distribution of the two EF-hands, fitted with double Gaussian curves (blue).
(D) The unfolding/refolding dynamics of the EF12 at a loading rate of ∼0.5 or −0.5 pN s−1(left), or at constant forces (right). The colored lines in the force-extension curves are 5- or 10-point FFT (fast Fourier transform) smoothing of the raw data (gray), if not specifically stated.
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4. Figure 2 Force-Dependent Stability and Kinetics of α-Actinin 1 SRs
(A) Left to right: typical force-extension curves of the rod construct, the double-rod construct, and SR23 construct, respectively, during the force-increase scan with a loading rate of ∼1 pN s−1. Each red arrow indicates the unfolding of a SR.
(B) The unfolding stepsize (left) and unfolding force distributions (middle) of the SRs (data from the rod and double-rod constructs) at a loading rate of ∼1 pN s−1. The blue line is the Gaussian or double-Gaussian curve fitting of the data. Right: the unfolding force distribution of the SR23 construct at a loading rate of ∼1 pN s−1.
(C) Left to right: typical force-extension curves of the corresponding constructs during the force-decrease scan with a loading rate of ∼−0.3 or −0.1 pN s−1. The red arrows indicate the refolding events.
(D) The refolding stepsize (left) and refolding force (middle) distributions of the SRs (data from the rod and double-rod constructs) at a loading rate of ∼−0.1 pN s−1. Right: the force-dependent unfolding and refolding transition rates obtained from the unfolding/refolding force distributions based on Arrhenius’ law.
(E) Left to right: typical unfolding/refolding dynamics of SRs at constant forces of 5.0 pN (left), 5.5 pN (middle), and 6.4 pN (right), respectively.
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5. Figure 3
Force-Dependent Unpairing of Dimerized Rod in a Zipper Force Geometry
(A) The double-rod construct was connected by a long flexible linker (182 aa, FH1 sequence) so that at low forces, the two rod domains would dimerize (paired) while at higher forces, the dimerized rods could be unpaired with a release of the looped FH1 region.
(B) Typical force-extension curves of the double-rod construct at a loading rate of ∼1 pN s−1 and followed by ∼−0.3 pN s−1. The eight black arrows indicate the typical unfolding of the eight SRs, and the purple arrow indicates the unpairing of the two rods before SRs unfolding. The upper inset shows the refolding of the eight SRs during a force-decrease scan between 8 to 2 pN (within the dotted box range). The lower inset shows the repairing of the two rods at a low force of ∼0.6 pN.
(C) Left: typical force-extension curves of the unparing of two rods during a force-increase scan up to 11 pN (before the unfolding of SRs). Right: the unpairing force distribution of the two rods with a loading rate of ∼1 pN s−1. The blue line is the Gaussian curve fitting of the data.
(D) Left: the constructs design for the in vivo FRAP experiments. Right: the fluorescence recovery time trace after photobleaching of wild-type α-actinin 1 (black) and the mutations lacking the SR2 and SR3 domain (ΔSR23, red), lacking the ABD (ΔABD, blue), lacking the EF-hands (ΔEF, magenta), EF-hands only (EF1234, cyan), and two rods (8SR, orange). The characteristic rate obtained by exponential fitting is ∼0.14 s−1, 1.0 s−1, 1.2 s−1, 1.1 s−1, 1.6 s−1, and 0.38 s−1, respectively, N ≥ 10.
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6. Figure 4
Forces on the Crosslinked α-Actinin 1 Dimer Based on the Force-Dependent Transition Rates of SRs
(A) Typical force-extension curves of the α-actinin 1 dimer with various pulling rates (red, 0.1 nm s−1; blue, 1 nm s−1; purple, 10 nm s−1; orange, 50 nm s−1) obtained by Gillespie kinetics simulation. The extension here is from the 248th residue from one monomer to the 248th residue of another (illustrated in sketch). The drops in force during constant pulling corresponds to the unfolding of SR1.
(B) The averaged peak forces (blue) and mean forces (red) versus the pulling rates, n = 30. The peak force denote the force where unfolding occurs. The mean forces denote the averaged forces between the two peak forces. n = 30.
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7. Figure 5
Force-Dependent High-Affinity Binding of Vinculin to αVBS in SR4
(A) Typical force-extension curves of the rod construct before (black) and after (red, blue) addition of VD1 at a loading rate of ∼1 pN s−1.
(B) Typical force-extension curves of the double-rod construct before (black) and after (red, blue, orange) addition of VD1 at a loading rate of ∼1 pN s−1. The numbers at right bottom indicate the number of SRs can be refolded. Before each force-increase unfolding scan, the tether was hold at low forces of ∼1–2 pN for 120 s to allow full refolding.
(C) Typical force-extension curves of the six repeats of SR4 construct before (black for force-increase scan, gray for force-decrease scan) and after (red, blue) addition of VD1 at a loading rate of ∼0.7 pN s−1.
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