Browsing by Subject "Vinculin"

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.

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.