Fig 2: DLC1 and RIAM directly compete for talin binding in cells.(A) Schematic of the LOVTRAP system. Illumination with blue light converts the LOV domain into the light state and displaces the dark-state binding Zdk tag and associated molecule (here, RIAM) from the mitochondria to enable adhesion binding. (B) Imaging of cells with a blue laser immediately displaces RIAM from a mitochondria localization (RIAM channel, magenta inset), leading to increased adhesion binding of RIAM and reduced adhesion binding of DLC1 (green inset and time series on right); the dynamics is changed after FAK inhibition. (C and D) Average time intensity curves of adhesions from 15 cells from three independent repeats for control and FAK inhibition, respectively. (E to H) Adhesion intensities (normalized to whole cell intensities) at the time of peak adhesion enrichment for RIAM, displayed as box plots, and changes for each cell from three independent repeats and >5 cells quantified per repeat. (I) Time for RIAM to reach peak intensity at adhesion. (J) Time for DLC1 to return to initial intensity before stimulation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, paired t test.

Fig 3: DLC1 and RIAM binding to talin is altered in vivo in heart disease.(A and B) Nanoindentation measurement of sections from WT and MLP knockout hearts. Sections (20-μm thick) of n = 3 hearts each were measured using a nanoindenter (tip radius = 49 μm, stiffness = 0.49 N/m, and indentation depth = 2 μm). (A) Stiffness map overlaid over the bright-field image. Empty squares indicate areas where the measurement failed, possibly due to uneven topography. (B) Quantification. ****P < 0.0001, unpaired t test after logarithmic transformation of the (log-normal distributed) data. (C to F) In situ proximity ligation (PL) assays were performed on sections from WT and MLP knockout hearts with DLC1 and talin (C) and RIAM and talin antibodies (D); n = 3 animals each. (E and F) Integrated intensity of proximity labels were normalized by the number of nuclei per image, and >5 images were quantified per heart. Data are presented as mean per animal, normalized by the mean of all images of the respective repeat. **P < 0.01, unpaired t test.

Fig 4: DLC1 and RIAM directly compete for talin binding in vitro.FP binding and competition experiments of purified talin R7R8 with DLC1 TBS, RIAM TBS1, and paxillin LD1 synthetic peptides. (A) Overlay of the crystal structures of talin 1 R8 (gray) in complex with DLC1 TBS (blue, pdb 5FZT) and RIAM TBS1 (orange, pdb 4W8P) peptides, showing that the binding site for the two peptides overlap. (B) FP binding curves of purified R7R8 with TBS-F (blue, Kd = 4.92 ± 0.76 μM), RIAM TBS1 (orange, Kd = 9.21 ± 2.16 μM), or paxillin LD1 [green, not determined (ND)]. For clarity, the fluorescein-labeled peptides are designated with a “-F” in the figure. (C) FP competition experiment of R7R8 complexed with DLC1 TBS-F in competition with unlabeled RIAM TBS1. Increasing amounts of unlabeled RIAM peptide outcompetes the labeled DLC1 TBS-F as seen by a decrease in polarization [orange, inhibitory constant (Ki) = 90 ± 48 μM]. The R7R8-DLC1 TBS-F complex could not be outcompeted with unlabeled paxillin LD1 (green). No changes in signal were observed when DLC1 TBS-F was titrated with unlabeled RIAM TBS1 peptide (black).

Fig 5: Model of stiffness-dependent competition of talin interactome.Stiffness-dependent integrin signaling leads to altered levels of FAK activation. On a healthy stiffness, DLC1 has a higher affinity for binding to the talin R8 domain compared to RIAM, possibly due to direct phosphorylation through FAK (48), and, thus, can modify the level of RhoA activity. In addition, FAK is predicted to phosphorylate talin, and the phosphorylation might further contribute to the regulation (64). On a fibrotic stiffness, lower FAK activity reduces DLC1 phosphorylation, and DLC1 is outcompeted by RIAM for talin R8 binding. Because of the importance of DLC1 for fine-tuning the levels of RhoA activity, this affects the cardiomyocyte maturity and disease progression.