Vinculin-mediated Mechanocoupling in Epithelial Sheet Expansion

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2020

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Abstract

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.

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Gates, Evan Michael (2020). Vinculin-mediated Mechanocoupling in Epithelial Sheet Expansion. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/20966.

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