Browsing by Author "Hoffman, Brenton D"

Important physiological processes, including migration, morphogenesis, and differentiation, and pathophysiological processes, including cancer and fibrosis, have been increasingly tied to cell’s abilities to sense mechanical stimuli from the extracellular matrix (ECM) and either generate or respond to mechanical loads in turn. Mechanical stimuli from the ECM is integrated at focal adhesions (FAs), a subcellular structure consisting of hundreds of interacting proteins that mediate physical connections between the ECM and force-generating actin cytoskeleton. At the molecular level, underlying this integration process is mechanotransduction, where the mechanical deformation of load-bearing proteins alters protein function to regulate signaling. This process is thought to expose cryptic protein binding domains that lead to the downstream recruitment and formation of mechanosensitive protein complexes. However, an incomplete understanding of mechanotransduction, and the relevant molecular players involved, prevents a mechanistic understanding of all mechanosensitive processes. In turn, this has hindered advancements in the development of therapies to combat mechanosensitive diseases, as well as efforts to manipulate cell response through the design of bio-instructive scaffolds in tissue engineering and regenerative medicine. To address this issue, the central goal of this dissertation is to develop and utilize molecular-scale tools to probe the role of molecular-scale forces on protein function and elucidate relevant molecular players in mechanotransduction. To date, available techniques for studying the role of molecular-scale forces on protein function remain technically challenging and low throughout. Thus, we sought to develop novel imaging- and biochemical-based assays that were capable of probing protein response to molecular tension within cellular contexts where both spatiotemporal control of cellular force generation and signaling networks were maintained. More specifically, we developed two separate assays that work in concert to first, characterize the specificity of the protein’s molecular tension-sensitive recruitment to FAs, and then unbiasedly uncover all molecular tension-sensitive protein interactions. We developed an imaging-based assay, termed Fluorescence Tension Co-localization (FTC), that integrates immunofluorescence labeling, molecular tension sensors, and machine learning to determine the sensitivity, specificity, and context-dependence of molecular tension sensitive protein recruitment mechanisms. When we applied FTC to study the mechanical linker protein, vinculin, we found constitutive and context specific molecular tension-sensitive protein recruitment mechanisms that varied with adhesion maturation. More specifically, we found that in immature FAs, vinculin tension specifically recruits integrin-associated proteins while in mature FAs, vinculin tension specifically recruits actin-associated proteins. We also developed a separate biochemical based assay, that integrates proximity-dependent biotin labeling techniques with biophysical knowledge of key residues required for protein loading to determine the mechanosensitive binding interactions of key FA proteins. Using streptavidin pulldown assays to isolate the interacting proteins, we found that vinculin forms proximal protein interactions with an FA protein, migfilin, that has not been previously identified as a vinculin binding partner. In summary, this dissertation focuses on developing novel molecular-scale assays for studying mechanosensitive protein recruitment and interaction mechanisms. Using these tools, we identified multifaceted tension sensitive protein recruitment mechanisms associated with vinculin during adhesion maturation, as well as identified a novel proximal binding partner, migfilin. Overall, this establishes the importance of molecular loads across single proteins in regulating other protein activity. Widespread use of these developed assays will help elucidate a more mechanistic understanding of mechanotransduction through the identification and study of relevant molecular players.

The extracellular environment is a key regulator of cell behavior, providing both biochemical factors and mechanical signals to influence the form and function of cells. The process by which cells sense and respond to environmental mechanical signals is often mediated through force-dependent changes in protein structure and function through a poorly understood process known as mechanotransduction. Towards elucidating the molecular processes underlying mechanosensitive regulation, molecular tension sensors (MTSs) have been created to measure forces experienced by specific proteins inside cells. However, an incomplete understanding of the effects of intracellular forces on fluorescent protein (FP) function within the context of MTs limits sensor application and interpretation. To advance our understanding of the molecular events mediating mechanotransduction, it is necessary to improve on existing approaches as well as to develop new technologies for probing mechanical consequences inside cells. In this dissertation we aim to address this limitation by creating a generalizable assay for probing the effects of cell-generated forces on FP function towards improving the use and interpretation of MTSs. Additionally, we describe the development of a new “synthetic” actin crosslinking sensor which leverages FP mechanosensitivity to provide new insights into mechanical processes inside cells.In our initial efforts, we focused on investigating the effects of cell generated forces on FP function within vinculin-based MTSs. We chose vinculin as our model system as vinculin is a well-studied mechanosensitive protein, known to play a critical role in force transmission inside cells. Additionally, the vinculin tension sensor (VinTS) has been extensively characterized, validated, and utilized in a broad array of applications. Leveraging the relationship between FRET measurements and fluorophore stoichiometry with vinculin MTSs, we developed a generalizable assay for evaluating changes in ensemble MTS measurements in terms of fluorophore contributions. Furthermore, we validated this new method on an extensive MTS data set containing over 2000 cells expressing vinculin sensors. Our analysis revealed that FP stoichiometry within VinTS was modulated significantly within individual focal adhesions (FAs) in an actomyosin-dependent manner, and that both load magnitude and load duration likely play a role. Additionally, we found that this force-mediated loss of FP function, or “mechanical quenching,” is a reversible process, consistent with nonequilibrium transitions in protein structure. To investigate FP mechanosensitivity further, we developed an engineered FRET-based actin crosslinking (ABD) sensor to serve as an improved experimental platform, within which FPs would be subjected to higher loads in a manner free of endogenous biochemical regulation. Within this new system, higher tensile loading and FP mechanical quenching was observed at dynamic actin networks. Furthermore, we found that FP mechanical quenching within these sensors was mediated by non-muscle myosin II (NMII) activity and appears to be reversible. In addition, we found that FPs exhibit different sensitivities to intracellular mechanical loads. To probe the molecular origins of ABD sensors loading within cells, we manipulated the organization and dynamics of actin structures by tuning substrate stiffness within engineered in vitro culture systems. Using this approach, we found that ABD sensors reported increased loads and FP mechanical quenching at dynamic actin networks in response to softer substrates. By coupling FRET-based MTSs with the tunability of in vitro culture models, we demonstrated the application of ABD sensors to probe changes in tensile loading in response to environmental mechanical cues. In summary, this dissertation describes the development of novel tools for studying the effects of intracellular forces on FP function within the context of FRET-based tension sensors. Using these tools, we found that FPs, like mechanosensitive signaling proteins, can undergo nonequilibrium transitions in response to cell-generated forces. Based on these observations, we propose that FP mechanical quenching within MTSs could potentially serve as an entirely new way to visualize and probe mechanical consequences within force-sensitive proteins. By exploiting the mechanosensitivity of FPs as a mechanical consequence, new insights into molecular force-sensitive processes inside cells may be obtained.

Mechanical forces are potent drivers of many biological processes. The form and function of many tissues depends on cells receiving the proper mechanical signals either from neighboring cells or from the underlying matrix. During development, dynamic tissue movements are driven by cell contractility and stem cell fate depends in large part to the mechanical forces they feel in their local surroundings. Conversely, aberrant mechanosensitive signaling is associated with the pathological progression of several disease states, such as cancer and atherosclerosis, for which effective treatments are scarce. As such, understanding how cells physically interact with and detect mechanical aspects of their microenvironment is critical to both understanding developmental processes and developing new treatments for disease.

Mechanical information from the microenvironment is converted into biochemical signals inside cells through molecular scale processes, collectively referred to as mechanotransduction. Many of the events associated with mechanosensitive signaling and mechanotransduction are mediated by force-dependent changes in protein structure and function. However, the lack of available tools to study these molecular scale processes in cells is currently preventing further progress. To address this need, the goals of this dissertation were to (1) improve upon and expand the capabilities of existing tools to visualize molecular forces and (2) develop novel methodologies to detect force-sensitive signaling events inside cells.

We began by focusing on the further development and improvement of one of the most critical tools to mechanobiological investigations: Förster Resonance Energy Transfer (FRET)-based molecular tension sensors. While these sensors have contributed greatly to our understanding of mechanobiology, the limited dynamic range and inability to specify the mechanical sensitivity of existing sensors has hindered their use in diverse cellular contexts. Through both experiments and modeling efforts, we developed a comprehensive biophysical understanding of molecular tension sensor function that enabled the creation of new sensors with predictable and tunable mechanical sensitivities. We used this knowledge to create a sensor optimized to study the ~1-6pN loads experienced by vinculin, a critical linker protein that plays an integral role in connecting cells, via focal adhesions (FAs), to the extracellular matrix (ECM). Using this optimized sensor enabled sensitive detection of changes in molecular loads across single cells and even within individual FA structures. We also expanded the capabilities of tension sensors to investigate the potentially distinct roles of protein force and protein extension in activating mechanosensitive signaling. Specifically, a trio of these new biosensors with distinct force- and extension-sensitivities revealed that an extension-based control paradigm underlies cellular control of vinculin loading.

Since these sensors uniquely provide insight into which molecules are physically engaged and could be participating in mechanically-based signaling, we chose to investigate which cytoskeletal structures mediate patterns of vinculin loading at multiple length scales within the cell. Specifically, we focused on two active, but distinct force generating machineries inside cells: stress fibers (SFs) and lamellipodial protrusions (LPs). By measuring vinculin tension in various mechanical and biochemical contexts, we found significant evidence for vinculin’s involvement in force transmission from both LP and SF structures. However, the distribution of loads across vinculin at the level of a single FA was dramatically different between these two distinct actin structures. Specifically, asymmetric distribution of vinculin load along individual FAs was an exclusive feature of SF-associated FAs. Subsequent experiments showed that formation and maintenance of these gradient loading profiles also depends on vinculin’s interactions with key binding partners, suggesting that both the magnitude as well as the pattern of vinculin loading within FAs are independently regulated by cells, and thus might serve distinct purposes in cellular mechanosensing.

Towards understanding the biochemical consequences of protein load at the molecular level, we developed an imaging-based technique to detect one of the events most often implicated in mechanically-based signal transduction: the formation of force-sensitive protein-protein interactions (PPIs). While these force sensitive interactions have been extensively documented in vitro, the extent to which they occur inside living cells is debated. This imaging-based technique, which we refer to as fluorescence-force co-localization (FFC), involves simultaneous FRET imaging of a FRET-based tension sensor to visualize protein loading and correlate this with the recruitment of other species to areas of high molecular loads. With vinculin as a prototypical example and a screen-based approach in mind, we used immunofluorescence to measure the relative enrichment of 20 other key FA proteins in areas of high vinculin tension. Factoring in what we previously learned about (1) the importance of actin architecture and (2) the well-established role of vinculin alone in controlling FA composition, we provide a multiparametric perspective on a potential mechanotransduction node associated with high vinculin loads. Focusing on the top five hits from this FFC screen, subsequent experiments revealed a genuine vinculin tension-dependent interaction with migfilin. While the involvement of both vinculin and migfilin in cardiac settings is a tempting line of future work, the work presented in this dissertation even more powerfully provides a proof-of-principle for the detection of force-sensitive PPIs in cells.

In total, the techniques developed in this dissertation enable detection of multiple molecular events associated with mechanotransduction inside cells. The improvement of FRET-based tension sensors as well as the ability to define their mechanical properties a priori should expedite investigations of molecular forces in diverse biological contexts. Additionally, the realization of force-dependent PPIs inside cells provided by the FFC screen constitutes a significant step towards uncovering mechanically-based signaling mechanisms inside cells. The more widespread application of these tools will undoubtedly fuel our understanding of mechanotransduction and could enable better control of cell behaviors in engineered tissues as well as the development of treatments for mechanosensitive diseases.

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.

The coordinated movement of groups of cells, called collective cell migration (CCM), plays important roles in many developmental, physiological, and pathological processes. During CCM, cells remain mechanically coupled to their neighbors, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to transmit and adapt to mechanical forces. However, the molecular mechanisms that underly these mechanosensitive processes remain poorly understood, hindering efforts to manipulate CCM for therapeutic or engineering purposes. To address this gap, this dissertation develops and applies a combination of experimental and modeling approaches to investigate molecular scale mechanosensitive processes. In the first part of this dissertation, we asked how mechanical forces and biochemical regulation interact to control mechanical coupling during CCM. We focused on the mechanical linker protein vinculin, which is known to mediate adhesion strengthening. Using a set of Förster resonance energy transfer (FRET)-based biosensors, we probed the mechanical function and biochemical regulation of vinculin, elucidating a switch that toggles both the activation and molecular loading of vinculin at cell adhesions. We found that the vinculin switch controlled both the speed and coordination of CCM, resulting in a covariation of these variables that suggested changes in adhesion-based friction. To bridge molecular and cellular measurements, we developed molecularly specific models of frictional forces at cell adhesions based on the force-sensitive bond dynamics of key proteins. In these models, increases in vinculin activation and loading produced increases in friction at adhesion structures, and this was due to the engagement of vinculin-actin catch bonding. Together, this work reveals how the biochemical regulation of a linker protein (vinculin) affects a cell-level mechanical property (adhesion-based friction) to control a multicellular behavior (CCM).

In the second part of this dissertation, we focused on how cells sense mechanical forces at the molecular scale. This is thought to occur by force-induced changes in the structure/function of proteins. However, how forces affect protein function inside cells remains poorly understood due to a lack of tools to probe this inside cells. Motivated by in vitro work showing that the mechanical loading of fluorescent proteins (FPs) causes a reversible switching of their fluorescence, we investigated if this phenomenon could be detected inside cells to directly visualize force-sensitive protein function. Using a mathematical model of FP mechanical switching, we developed a framework to detect it inside FRET-based biosensors. Applying this framework, we observed FP mechanical switching in two sensors, a synthetic actin-crosslinker and the linker protein vinculin, and we found that mechanical switching was altered by manipulations to cellular forces on the sensor as well as force-dependent bond dynamics of the sensor. Together, this work develops a new framework for assessing the mechanical stability of FPs and enables visualizing the effect of forces on protein function inside cells.

Overall, the work in this dissertation advances our basic understanding of mechanosensitive processes, addressing knowledge gaps in CCM and mechanobiology. The frameworks we have developed for integrating molecular- and cellular-level experiments with mathematical models will facilitate new mechanistic studies into mechanosensitive processes involving other proteins and biological contexts.

Bacterial cell division is tightly coupled to the dynamic behavior of FtsZ, a tubulin homolog. Recent experimental work \textit{in vitro} and \textit{in vivo} has attributed FtsZ’s assembly dynamics to treadmilling, where subunits add to the bottom and dissociate from the top of protofilaments. However, the molecular mechanisms producing treadmilling have yet to be characterized and quantified. We have developed a Monte Carlo model for FtsZ assembly that explains treadmilling and assembly nucleation by the same mechanisms. A key element of the model is a conformational change from R (relaxed), which is highly favored for monomers, to T (tense), which is favored for subunits in a protofilament. This model was created in MATLAB. Kinetic parameters were converted to probabilities of execution during a single, small time step. These were used to stochastically determine FtsZ dynamics. Our model is able to accurately describe the results of several \textit{in vitro} and \textit{in vivo} studies for a variety of FtsZ flavors. With standard conditions, the model FtsZ polymerized and produced protofilaments that treadmilled at 24 nm/s, hydrolyzed GTP at 2.4 to 3.2 GTP min\textsuperscript{-1} FtsZ\textsuperscript{-1}, and had an average length of 30 to 60 subunits, all similar to experimental results. Adding a bottom capper resulted in shorter protofilaments and higher GTPase, similar to the effect of the known the bottom capper protein MciZ. The model could match nucleation kinetics of several flavors of FtsZ using the same parameters as treadmilling and varying only the R to T transition of monomers.

Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).

Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.

The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.

For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.

The extracellular environment affects tissue structure and is a source of physical cues to guide cell behavior. The sensing and interpretation of physical cues is important in many physiological processes, such as tissue organization and cell differentiation in development. Additionally, the misregulation or misinterpretation of physical cues is implicated in a variety of disease states that are currently lacking in effective treatments, including cancer metastasis and vascular disease. The process by which cells sense and respond to the physical nature of their environment is termed mechanotransduction and is mediated by molecular-scale protein-protein interactions that enable transmission of forces between the extracellular- and intracellular-environment for subsequent interpretation. Progress in understanding the molecular mechanisms important in mechanotransduction has been hindered by the lack of tools and analyses specifically designed for measuring the mechanical state of proteins within mechanosensitive structures in living cells. To address this issue, the goals of this dissertation were to: 1) create novel tools for measuring mechanical state of a protein, 2) investigate the role of protein mechanical state in mediating cellular response to physical cues.

We first aimed to create a technique for measuring force-sensitive protein dynamics within living cells, a property that had previously only been accessible in a purified, in vitro, system that does not properly mimic the cellular context. We chose to work with the mechanical linker protein vinculin, which is implicated in transducing forces at the site of cell-extracellular matrix (ECM) interactions, termed focal adhesions (FAs). By combining a Förster Resonance Energy Transfer (FRET)-based sensor to measure forces across vinculin with Fluorescence Recover After Photobleaching (FRAP) to measure vinculin dynamics, we were able to directly probe vinculin force-sensitive dynamics at FAs in living cells for the first time. The results allowed us to discover three distinct vinculin states: a force-stabilized state, a force-destabilized state, and a force-insensitive state. These states depend on cytoskeletal regulation and the ability of vinculin to bind critical binding partners. We found that the force-stabilized state of vinculin is required to concurrently stabilize FAs and cell protrusions. While the force-stabilized state and force-destabilized state can both mediate 2D random migration, when cells are presented with a physical gradient of ECM across a 3D pore, only the vinculin force-stabilized state is permissive for cells to successfully migrate in response to the physical cue.

To investigate the role of vinculin in sensing a different physical cue, we chose to confine cells to micropatterns of the same area but different shape and probe the effect on vinculin mechanical state. We again used cells expressing the FRET-based vinculin tension sensor (VinTS), as well as a force-insensitive control (VinTL), to measure the spatial regulation of vinculin loading in response to cell shape. It was shown that cell confinement leads to a gradient in vinculin load, with high tension at the edges and corners of cells, and a novel state at the center of cells that was potentially consistent with previous observations of compressive forces in this region. The incidence of vinculin compression was increased with increased aspect ratio of patterns, meant to induce higher contractility states. Additionally, different forms of cytoskeletal inhibition led to distinct modulations of the spatial distribution of vinculin load, suggesting different mechanisms for regulating vinculin tension and compression. We concluded that confining cells to micropatterns led to actin organization above the nucleus, resulting in compression of the nucleus, which was transferred to the FAs below, generating a spatial gradient of vinculin loading.

In continuing to study the role of the mechanically distinct FAs in the center of confined cells in mechanotransduction, we chose to focus on the biophysical and biochemical regulation, structure, and composition of confinement-induced central FAs. First, we determined that the uniqueness of this state was at least partially due to dimerization or clustering of vinculin at central FAs, suggesting a different structural organization from peripheral FAs. We used different degrees of confinement to show that increased confinement leads to increased incidence of central FAs, but we were unable to induce the formation of these FAs through cytoskeletal manipulation in the absence of physical cues. To understand how these central FAs might be uniquely regulating cell response to confinement, we identified a protein only recently discovered to localize to FAs that is enriched in our central FAs, called KANK2. Through knockdown of KANK2, we found that KANK2 regulates vinculin expression and localization and is required for the formation of central FAs. We concluded that the interaction of KANK2 with key FA proteins, including vinculin, regulates cell sensing and response to physical confinement, suggesting an independent pathway from the regulation of the highly studied peripheral FAs. Determining if this novel pathway can be used to affect cell behavior is an interesting future application of this work.

In summary, this dissertation describes novel tools for studying mechanical states of proteins within living cells and demonstrates the importance of protein mechanical state in mediating cell response to physical cues, using the FA mechanical linker protein vinculin as a prototypical example. We find that vinculin is a critical molecular player in allowing cells to sense and respond to haptotactic cues and cell confinement. We expect that future work in studying cell response to other physical cues, for example substrate stiffness, or in different subcellular structures such as adherens junctions (AJs) will continue to implicate vinculin as having an important role in mechanotransduction. Additionally, we believe that the new tools, assays, and analyses that we have developed will contribute to the study of molecular mechanisms necessary for mechanotransduction in different proteins, subcellular structures, and cellular contexts. Understanding how cells sense and respond to physical cues in guiding cell behavior will aid in the creation of therapies for mechanosensitive disease states as well as provide means for controlling cell behavior for tissue engineering or regenerative applications.

Cell migration and multicellular interactions are essential for the formation and maintenance of tissue structure. The dysregulation of these processes also contributes to developmental defects and pathological processes. A prominent question is how biochemical and biophysical information, which acts at the level of an individual cell, is transmitted and integrated by neighboring cells to yield coordinated behavior. In a process known as collective cell migration (CCM), mechanical coupling of cells is thought to play a key role in coordinating migration across many cell lengths. Mechanocoupling refers to the mechanical integration of cell-cell adhesions and the contractile actomyosin network. While pertinent signaling pathways have been identified that mediate CCM, the mechanisms involved in mechanocoupling at the molecular level are poorly understood. Progress in the field has been limited due to the molecular complexity of adhesion structures and technical limitations of measuring in vivo mechanics to identify mechanosensitive elements. Therefore, a central but understudied phenomenon in cell migration is the study of mechanocoupling. The overall premise of this proposal is that we can use a new type of force-sensitive biosensor to identify proteins responsible for mediating mechanocoupling. The advances from this approach will fundamentally advance our understanding of CCM and open new doors for the manipulation and control of CCM.

The force-sensitive biosensor used in this work was a Fӧrster resonance energy transfer (FRET)-based tension sensor, which enables the measurement of molecular-scale forces across proteins based on changes in emitted light. We focused specifically on the role of vinculin in mediating mechanocoupling for two important reasons. Firstly, vinculin is the only protein known to localize to both FAs and AJs in response to mechanical loading. Secondly, vinculin activity can be regulated by multiple kinases through site-specific phosphorylation. However, the implications of vinculin regulation by these kinases has not been fully elucidated. As the reliability and reproducibility of measurements made with FRET-based tension sensors has not been thoroughly examined, we first developed numerical methods that improve the accuracy of measurements made using sensitized emission-based imaging. To establish that FRET-based tension sensors are versatile tools that provide consistent measurements, we then used these methods to demonstrate that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggested FRET-based tension sensors could be coupled with a variety of immuno-fluorescent labeling techniques for future investigations into mechanocoupling. Additionally, as tension sensors are frequently employed in complex biological samples where large experimental repeats may be challenging, we examined how sample size affects the uncertainty of FRET measurements. In total, this groundwork established useful guidelines to ensure precise and reproducible measurements for studying mechanics in CCM using FRET-based tension sensors.

To investigate the mediators of mechanocoupling in CCM, epithelial sheet migration was studied because it is characterized by long-range coordination and, presumably, high mechanocoupling. Two epithelial cell lines were subjected to a non-wounding 2D migration assay and found to exhibit stark differences in migratory characteristics, including speed and velocity correlations. The pertinent subcellular structures for mechanocoupling, namely focal adhesions (FAs), adherens junctions (AJs), and the actomyosin cytoskeleton, appeared to contribute to these differences. A significant finding was that actin belts, traditionally associated with long-range coupling in developmental events, did not lead to global coordination within a migrating layer. Instead, measurements of vinculin tension demonstrated that vinculin mechanocoupling was associated with long-range coordination throughout a migrating layer and the formation of a pluricellular actin network. Interestingly, vinculin was shown to act as a mechanocoupler throughout a cell’s cytoplasmic actin network, demonstrating a previously unappreciated role of vinculin. Universally, vinculin mechanocoupling involved actin interactions and required a head-specific site known to interact with a variety of binding partners including talin, β-catenin, α-catenin, and α-actinin. As vinculin can undergo head-tail autoinhibition, its conformation was evaluated. These findings indicated that vinculin was differentially regulated. By probing the role of three kinases, it was found that serine phosphorylation by Protein Kinase C (PKC) is an important regulator of vinculin mechanocoupling.

In summary, we propose that long-range coordination during CCM can be mediated by mechanocoupling of a supracellular actin network. Based on our findings, vinculin mechanocoupling is associated with the emergence of this supracellular network. Furthermore, serine phosphorylation appears to play a previously underappreciated role in regulating the mechanical integration of migrating cells. These advancements serve as an important step toward better understanding the physical mechanisms of CCM.