Elucidation of the Role of the Vinculin-Actin Catch Bond in Fibroblast-Mechanical Microenvironment Feedback

Abstract

Fibroblasts resist or adapt to mechanical forces in response to their mechanical microenvironment1,2 via load-bearing proteins maintaining or losing connectivity in response to these forces. At protein-protein interfaces, one mode of force-dependent biological adhesion control is enabled by catch bonds3,4, which counterintuitively strengthen in response to applied force. Catch bonds exhibiting directional asymmetry have been identified at cell adhesion substructures5,6 and are predicted to have key roles in polarized molecular and cellular processes5. However, experimental confirmation is currently lacking, as there no approaches exist to validate and characterize catch bond behavior inside cells. To address this issue, the goals of this dissertation are to 1) create novel tools to characterize a molecular mechanism of catch bonding in cells and 2) investigate the role of catch bonding in key subcellular and cellular processes at varying length and time scales. We chose to focus on the mechanical linker protein vinculin, as it is known to mediate force transmission in between the actin cytoskeleton and the extracellular matrix (ECM) and to form the strongest single interface catch bond characterized to date with F-actin in a directionally asymmetric manner5. With the aid of the Campbell lab at the University of North Carolina-Chapel Hill, the identities of the two amino acids mediating directionally asymmetric force strengthened (DAFS) interactions between vinculin and actin were determined using discrete molecular dynamics simulations (E1015, E1021). Furthermore, alanine mutations to DAFS residues were found to conserve vinculin structure and actin binding ability, ensuring that DAFS variants conserved force-independent vinculin functions. In the first part of this thesis, we assessed whether DAFS residues in vinculin mediate vinculin:F-actin catch bonding and used a systematic approach of incorporating single and double DAFS variants in vinculin to gradually perturb force-strengthening. We observed that all DAFS variants conserved the force-independent vinculin biological functions of localization and activation. We then probed vinculin loading using vinculin tension sensors7-11 (VcnTSs) and found the double DAFS variant showed the most significant decrease in vinculin load short of unloading. The support of an intermediate level of loading is expected for loss of catch bonding but not loss of vinculin:F-actin interactions. Thus, these data show that DAFS residues contribute to vinculin loading and are consistent with DAFS residues mediating force-strengthening behavior in vinculin. We then used fluorescence recovery after photobleaching (FRAP) to characterize changes in vinculin dynamics and found DAFS variants exhibited increased mobility, indicating a decreased stably adhered population at the FA. Assessing the relationship between vinculin load and vinculin dynamics using the FRET-FRAP technique9,12, we discovered that vinculin’s native tension-stabilized state is ablated with the introduction of DAFS variants in vinculin tension sensor. This loss of tension-stabilized behavior is consistent with catch bond behavior disruption9. Altogether, these data indicate that the inclusion of DAFS variants E1015A and E1021A in vinculin prevents tension-stabilized exchange dynamics, consistent with predictions of loss of vinculin:F-actin catch bonding in cellulo13. Next, we assessed the functional consequences of perturbing vinculin:F-actin catch bonding by characterizing its role in protein recruitment and fibroblast processes at different timescales. We first assessed vinculin tension-sensitive protein recruitment, a process that occurs on the timescale of subseconds at the molecular level. We chose to assess the tension-sensitive recruitment of migfilin, a LIM domain protein previously found to localize to focal adhesions with high vinculin tension11. We found that migfilin recruitment is promoted by the engagement of the vinculin:F-actin catch bond. We further demonstrated that vinculin:F-actin catch bonding affects the short-term, force-sensitive process of adhesion strengthening. Using machine learning to identify mature, stress fiber-associated FAs11, we discovered that the engagement of DAFS residues enables higher spatial variation in load at stress fiber-associated FAs. A functional consequence of this is greater local FA-stress fiber alignment, which we assessed using a novel method to relate FA orientation to local regions of stress fibers. We then probed the medium-term force sensitive response of substrate stiffness sensing in WT VcnTS and vinculin:F-actin catch bonding deficient VcnTS on soft (3.5 kPa), medium (12 kPa) and glass substrates, using YAP nuclear translocation. We found that across the range of stiffnesses probed, YAP was able to translocate to the nucleus for WT VcnTS expressing MEFs and that vinculin:F-actin catch bonding deficient cells showed decreased YAP nuclear translocation on the soft gel. Thus, vinculin:F-actin catch bonding plays an important role in stiffness sensing, especially at physiologically relevant stiffnesses. We further demonstrated that vinculin:F-actin directionally asymmetric catch bonding is important for another medium-term force sensitive process by conducting a Boyden chamber haptotaxis9,14 assay on WT and vinculin:F-actin catch bonding deficient cells. We discovered that the vinculin:F-actin catch bonding deficient cells showed decreased number of migrated cells, indicating engagement of the vinculin:F-actin catch bond promotes sensing of ECM gradient cues. Lastly, we assessed the role of vinculin:F-actin catch bonding in collagen compaction and FMT, two long-term force-sensitive processes. We found that both WT and DAFS double variant VcnTS underwent collagen compaction and that vinculin is not necessary for fibroblast to myofibroblast transition. Thus, at long-term responses on the order of days to weeks, we found that vinculin:F-actin catch bonding did not play a significant role. In summary, this dissertation presents a paradigm for evaluating catch bond behavior in cells using molecular mechanobiology tools and assays. This work also demonstrates the importance of the vinculin:F-actin catch bond specifically in mediating cellular processes at different timescales an higher length scale mechanobiology. We believe that the tools, assays, and analyses to characterize the functional role of the vinculin:F-actin catch bond can contribute to the study of other relevant catch bonds at cellular adhesion structures and across molecular biology. For example, the vinculin:F-actin catch bond residues bear homology to α-catenin: F-actin catch bond residues. Future studies validating and characterizing the in cellulo role of α -catenin: F-actin catch bonding could be conducted to better understand collective cell migration and morphogenesis. Ultimately, the paradigm to study force-sensitive bond dynamics inside cells will aid in the creation of studies to evaluate the role of force-sensitive bond dynamics in key biological processes.