Browsing by Subject "mechanotransduction"
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Item Open Access Biomaterials-Mediated Regulation of Macrophage Cell Fate.(Frontiers in bioengineering and biotechnology, 2020-01) Liu, Yining; Segura, TatianaEndogenous regeneration aims to rebuild and reinstate tissue function through enlisting natural self-repairing processes. Promoting endogenous regeneration by reducing tissue-damaging inflammatory responses while reinforcing self-resolving inflammatory processes is gaining popularity. In this approach, the immune system is recruited as the principal player to deposit a pro-reparative matrix and secrete pro-regenerative cytokines and growth factors. The natural wound healing cascade involves many immune system players (neutrophils, macrophages, T cells, B cells, etc.) that are likely to play important and indispensable roles in endogenous regeneration. These cells support both the innate and adaptive arms of the immune system and collectively orchestrate host responses to tissue damage. As the early responders during the innate immune response, macrophages have been studied for decades in the context of inflammatory and foreign body responses and were often considered a cell type to be avoided. The view on macrophages has evolved and it is now understood that macrophages should be directly engaged, and their phenotype modulated, to guide the timely transition of the immune response and reparative environment. One way to achieve this is to design immunomodulating biomaterials that can be placed where endogenous regeneration is desired and actively direct macrophage polarization. Upon encountering these biomaterials, macrophages are trained to perform more pro-regenerative roles and generate the appropriate environment for later stages of regeneration since they bridge the innate immune response and the adaptive immune response. This new design paradigm necessitates the understanding of how material design elicits differential macrophage phenotype activation. This review is focused on the macrophage-material interaction and how to engineer biomaterials to steer macrophage phenotypes for better tissue regeneration.Item Embargo Experimental and Modeling Approaches to Investigate Molecular-Scale Mechanosensitive Processes in Collective Cell Migration(2024) Shoyer, Timothy CurtisThe 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.
Item Open Access Lysophosphatidic Acid Induces ECM Production via Activation of the Mechanosensitive YAP/TAZ Transcriptional Pathway in Trabecular Meshwork Cells.(Investigative ophthalmology & visual science, 2018-04) Ho, Leona TY; Skiba, Nikolai; Ullmer, Christoph; Rao, Ponugoti VasanthaLysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance to aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of the TM to LPA, however, is not completely understood. In this study, we explored the possible involvement of mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), transcriptional activation in extracellular matrix (ECM) production by LPA-induced contractile activity in human TM cells (HTM).The responsiveness of genes encoding LPA receptors (LPARs), LPA hydrolyzing lipid phosphate phosphatases (LPPs), and the LPA-generating autotaxin (ATX) to cyclic mechanical stretch in HTM cells, was evaluated by RT-quantitative (q)PCR. The effects of LPA and LPA receptor antagonists on actomyosin contractile activity, activation of YAP/TAZ, and levels of connective tissue growth factor (CTGF), and Cyr61 and ECM proteins in HTM cells were determined by immunoblotting, mass spectrometry, and immunofluorescence analyses.Cyclic mechanical stretch significantly increased the expression of several types of LPARs, LPP1, and ATX in HTM cells. LPA and LPA receptor-dependent contractile activity led to increases in both, the protein levels and activation of YAP/TAZ, and increased the levels of CTGF, Cyr61, α-smooth muscle actin (α-SMA), and ECM proteins in HTM cells.The results of this study reveal that LPA and its receptors stimulate YAP/TAZ transcriptional activity in HTM cells by modulating cellular contractile tension, and augment expression of CTGF that in turn leads to increased production of ECM. Therefore, YAP/TAZ-induced increases in CTGF and ECM production could be an important molecular mechanism underlying LPA-induced resistance to AH outflow and ocular hypertension.Item Open Access Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.(Proc Natl Acad Sci U S A, 2014-11-25) Lee, Whasil; Leddy, Holly A; Chen, Yong; Lee, Suk Hee; Zelenski, Nicole A; McNulty, Amy L; Wu, Jason; Beicker, Kellie N; Coles, Jeffrey; Zauscher, Stefan; Grandl, Jörg; Sachs, Frederick; Guilak, Farshid; Liedtke, Wolfgang BDiarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca(2+) signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca(2+) transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.