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. 2016 Jul 26;10(7):6648-58.
doi: 10.1021/acsnano.6b01658. Epub 2016 Jul 11.

All Subdomains of the Talin Rod Are Mechanically Vulnerable and May Contribute To Cellular Mechanosensing

Alexander William M Haining  1 , Magdaléna von Essen  2   3 , Simon J Attwood  1 , Vesa P Hytönen  2   3 , Armando Del Río Hernández  1
Affiliations

All Subdomains of the Talin Rod Are Mechanically Vulnerable and May Contribute To Cellular Mechanosensing

Alexander William M Haining et al. ACS Nano. .

Abstract

Although the relevance of mechanotransduction in cell signaling is currently appreciated, the mechanisms that drive this process remain largely unknown. Mechanical unfolding of proteins may trigger distinct downstream signals in cells, providing a mechanism for cellular mechanotransduction. Force-induced unfolding of talin, a prominent focal adhesion protein, has been demonstrated previously for a small portion of its rod domain. Here, using single-molecule atomic force microscopy (smAFM), we show that the entire talin rod can be unfolded by mechanical extension, over a physiological range of forces between 10 and 40 pN. We also demonstrate, through a combination of smAFM and steered molecular dynamics, that the different bundles within the talin rod exhibit a distinct hierarchy of mechanical stability. These results provide a mechanism by which different force conditions within the cell control a graduated unfolding of the talin rod. Mechanical unfolding of the rod subdomains, and the subsequent effect on talin's binding interactions, would allow for a finely tuned cellular response to internally or externally applied forces.

Keywords: mechanobiology; mechanotransduction; protein mechanostability; single-molecule force spectroscopy; steered molecular dynamics.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Talin rod polyprotein constructs for smAFM experiments. (a) Crystal structure of talin rod, showing the α-helical bundles R1–R12. The rod was separated into different fragments as indicated for the AFM experiments. (b) Schematic of the polyprotein constructs containing: the talin fragment to be investigated; four flanking I27 domains for fingerprinting; the HaloTag enzyme that binds to its associated ligand on the surfaces; a HisTag for protein purification; and a terminal cysteine for cantilever tip attachment. (c) Example of fingerprint used for identification. After the unfolding of the talin rod fragment, the first peak corresponds to the unfolding of the HaloTag enzyme. This is followed by the unfolding events for the four I27 domains, creating the characteristic “sawtooth” pattern. The final peak represents the detachment of the polyprotein from either the surface or the cantilever. The dashed lines indicate a worm-like chain model fit applied to the I27 unfolding events. (b, c) Not to scale.
Figure 2
Figure 2
Unfolding pattern of talin rod fragments. (a–e) Aggregated force extension traces reveal the unfolding pattern of each fragment. The fingerprints of each trace were used for alignment, revealing the number of unfolding events and the extension associated with each. Inset: crystal structures for each fragment. Each event is fitted to the worm-like chain model (dashed lines). The method for measuring the extension of each event is also shown. (f–j) Histograms of the extensions associated with each unfolding events. The extension associated with each unfolding event was measured, using HaloTag as a reference point. The x-axes have been reversed so that the order of the unfolding peaks in the histogram matches the order of unfolding in the traces, i.e., the furthest peak from HaloTag is the first to unfold. Red dashed lines show Gaussian fits applied to the histograms. Extension lengths are summarized in Supporting Information Table S1. (a, f) R1–R3, number of traces analyzed, n = 101. (b, g) R4–R6, n = 76. (c, h) R7–R8, n = 82. (d, (i) R9–R10, n = 91. (e, j) R11–R12, n = 103.
Figure 3
Figure 3
Unfolding force of talin rod fragments using smAFM. (a–e) Histograms of the unfolding force for each event. Gray bars represent the unfolding event closest to HaloTag. Blue bars, where applicable, show the next event further from HaloTag. Finally green bars show the first unfolding event, and furthest from HaloTag, for those traces exhibiting three events. Red dashed lines show Gaussian fits applied to the histograms. Forces are summarized in Supporting Information Table S1. (a) R1–R3. (b) R4–R6. (c) R7–R8. (d) R9–R10. (e) R11–R12.
Figure 4
Figure 4
Unfolding force magnitude (pN) of talin rod domains in SMD. (a–c) Force against simulation time during constant velocity pulling at 2 nm/ns. (d–f) Force against simulation time during constant velocity pulling at 0.1 nm/ns. Gray lines visualize the force magnitude for all bundles. The tested rod domains show varying mechanical stability represented by different unfolding force magnitude. All rod domains are classified into three groups which are color highlighted in separate plots against the complete data set. (a, d) Weak class (orange) contains R3, R4, R6, and R10 bundles unfolding under maximum unfolding force 500pN; (b, e) intermediate class (blue) contains bundles R7, R11, and R12 unfolding between 500 and 1000pN; (c, f) strong class (green) with R5 and R9 bundles with unfolding force over 1000 pN. Similar classification was observed for both simulation settings at 0.1 nm/ns and 2 nm/ns velocity.
Figure 5
Figure 5
Representative structure snapshots of talin rod domains. Structure snaps were captured at 0, 5, 10, and 15 ns of the constant velocity pulling at 2 nm/ns. The three stability groups (weak, orange; intermediate, blue; strong, green) show mild yet distinct differences in the domain unfolding. Fully unfolded helices are cut away and presented by dashed line with a helix identifier.
Figure 6
Figure 6
Cellular mechanotransduction as a result of graduated talin rod unfolding. Without force, the talin rod remains fully structured, and no VBSs are available. Under low-force regimes, only the very weakest bundle, R3, unfolds revealing its VBS. This activates one vinculin molecule, releasing it from its autoinhibited state. As the force applied to talin increases, more bundles are unfolded, revealing more VBSs and thus activating an increasing number of vinculin molecules. The helical bundles have been colored to reflect the mechanical hierarchy shown in Figure 4. This schematic represents a simplified version of vinculin-mediated talin mechanosensitivity. The process in vivo is likely to include additional binding partners and more complex modes of force application and unfolding.
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