Browsing by Subject "Mechanotransduction"

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.

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.

Mechanosensation, or the ability to sense mechanical forces, is critical for the survival of all organisms. For vertebrates, the ability to sense and respond to environmental stimuli, or somatosensation, is not well understood at the neural circuit level, and the molecular underpinnings for somatosensation, especially in regards to mechanosensation, remain elusive and unresolved. In recent years, Piezo ion channels were discovered as the first mammalian excitatory bona fide mechanosensitive ion channel to be the initial molecules in sensing gentle touch in the somatosensory neural circuitry. Although physiological roles of Piezo ion channels for both somatosensation and other non-neuronal processes have been identified, the mechanisms for how these ion channels directly sense mechanical forces and transduce electrical signals remains unknown.

With the use of biochemical, molecular, and electrophysiological methods, I first developed a novel technique in which electrophysiology could be performed on microsomes, or vesicles formed from ER fractions containing proteins that are not trafficked to the plasma membrane. This experiment revealed that wild-type Piezo1 ion channels retain stretch activation in microsomes, and therefore this technique could be utilized to characterize mutants channels that lack trafficking to the plasma membrane in order to identify which domains are involved in activation and inactivation of Piezo ion channels. I next generated two separate constructs that removed domains suggested to be involved in activation and inactivation of Piezo1. Removal of the proposed inactivation domain rendered a non-functional channel that could still trimerize, suggesting that this domain not only plays a role in inactivation but also is critical for activation of Piezo1 ion channels. Partial removal of the proposed membrane-spanning mechanosensor domains produced a channel that lacked the ability to conduct large macroscopic currents but formed a conducting pore with single channel openings. This finding suggests that membrane tension is not sensed and transduced to the pore by a single domain, but rather multiple domains in concert. Together the findings provide evidence that the mechanisms for both activation and inactivation require multiple domains moving in collaboration together, and will broadly be informative for continued studies of the molecular mechanisms of mechanosensitive ion channels.

An organism’s ability to sense mechanical forces is critical for the detection of environmental stimuli as well as the regulation of internal processes necessary for survival. In vertebrates, the molecular mechanisms of somatosensation has remained an important and unresolved neurobiological question. In the past decade, Piezo ion channels have emerged as the first vertebrate ion channels identified responsible for transforming somatosensory stimuli into excitatory signals in the nervous system, generating our sense of touch. However, little still is known about the precise mechanisms for how Piezo channels activate and inactivate in the presence of stimuli and how they could potentially be modulated.

By using a combination of engineered and biomolecular methods combined with electrophysiology, I identify distinct structural domains within Piezo ion channels that are necessary for specific aspects of channel inactivation. First, I engineered a method by which magnetic nanoparticles were used to mechanically pull on individual domains of the Piezo channel to screen for mechanically sensitive structures. This experiment revealed a particularly striking effect for one domain, manifested as a profound slowing of channel inactivation kinetics. Next, I generated chimeric constructs to exchange this domain with a homologous structure and demonstrated its sufficiency for mediating the kinetics for inactivation. Finally, I introduced point mutations at key residues within the immediately adjacent pore domain and identified a structural correlate for the modulation of inactivation by voltage. These findings together provide a foundation for understanding the mechanism for inactivation in Piezo channels, and more broadly, for further studying the complexities in transducing mechanical force that create our sense of touch.

The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable engineered skeletal muscle tissue. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.

We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.

We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a more simple, 2D system. In particular, we assessed miRNA and muscle-specific gene expression over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as SRF and Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response and suggest that a balancing mechanism is in place to avoid large fluctuations in miRNA levels.

Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, technological limitations have precluded a detailed quantitative understanding of cells as sensors of mechanical energy. Here, I describe the development and validation of a system combining atomic force microscopy with patch-clamp electrophysiology. Using this system, I develop a quantitative framework for describing cells as sensors of mechanical energy and determine the physical limits of detection and resolution of mechanical energy for cells expressing the force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. I find that, depending on the identity of the channel, cells can function either as proportional or nonlinear transducers of mechanical energy, detect mechanical energies as little as ~70 fJ, and with a resolution of up to ~1 fJ. I also make the surprising discovery that cells can transduce forces either nearly instantaneously (< 1ms), or with substantial time delay (~10 ms) dependent on the identity of the channel. Using a chimeric experimental approach and simulations we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in cellular membranes. Further, I explore the role that cellular properties such as cell size, channel density, and actin cytoarchitecture play in tuning the biophysical limits of rapid cellular mechanotransduction. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

The meniscus of the knee is a fibrocartilaginous structure essential to the biomechanical integrity and function of the knee joint. Millions of people suffer meniscus injuries each year, and meniscus tears are common at all ages and stages of life. Meniscus injury has long-term consequences: loss of meniscus function has been definitively linked to early-onset osteoarthritis. Treatment options for meniscus injury remain limited; although there has been a recent movement to surgically repair the meniscus whenever possible, partial meniscectomy remains one of the most commonly performed orthopedic surgeries. Due to this urgent need, there is currently great interest in methods to stimulate meniscus healing, augment repair, and improve long-term outcomes following meniscus injury using pharmaceutical, biological, or tissue engineering methods. Research on meniscus regenerative medicine, however, is greatly limited by a lack of understanding of meniscus cellular biology.In this dissertation, this gap in knowledge is addressed with a thorough characterization of the meniscus cell phenotype. We have investigated the phenotypic identity of meniscus cells from inner and outer zones of the meniscus by RNA-sequencing, and provide comparisons to articular cartilage and isolated, monolayer cultured meniscus cells. We found that in situ meniscus cells from both the inner and outer zones are strikingly distinct from either articular chondrocytes or monolayer expanded meniscus cells at the transcriptomic level, and that inner and outer zone meniscus cells may be more similar to each other than to chondrocytes or monolayer cultured cells. Differences were also observed between inner and outer zone cells, and this dataset provides a wealth of novel targets for characterizing inner and outer zone cells to better understand regional cell biology of the meniscus. We investigated the role of the physical microenvironment, including native extracellular matrix, monolayer culture, and biomaterial hydrogels, to modulate the meniscus cell transcriptional phenotype and support meniscus cell culture and expansion in vitro. Our findings provide new details on the meniscus cell dedifferentiation process, and demonstrate the utility of bioengineered hydrogels to reverse meniscus cell dedifferentiation for long-term in vitro culture. We also investigated the effect of an injury-relevant inflammatory stimulus (IL-1), and the potential for dynamic mechanical loading to modulate the inflammatory response of meniscus cells in two models of dynamic physiologic loading, cell stretch of isolated meniscus cells and compression of tissue explants. Results of RNA-sequencing, gene set enrichment analysis, and RT-qPCR from both models showed significant modulation of inflammation-related genes and pathways with mechanical stimulation, supporting the potential of mechanotransduction pathways as novel therapeutic targets to improve outcomes following meniscus injury. Overall, this work provides a wealth of data characterizing the meniscus cell phenotype and lays the groundwork for future studies of meniscus regenerative medicine and tissue engineering. Furthermore, this work entailed considerable development and validation of methods for in vitro studies of meniscus cell biology and mechanotransduction, which will be valuable to the field of meniscus research. The work presented in this dissertation represents an enormous step forward in understanding the effects of physical, mechanical, and inflammatory environments on the meniscus cell phenotype, which is essential to the development of effective novel therapies to stimulate meniscus repair and prevent PTOA.

Physical forces play a key role in the morphogenesis of embryos. As cells and tissues change shape, grow, and migrate, they exert and respond to forces via mechanosensitive proteins and protein complexes. How the response to force is regulated is not completely understood.

Dorsal closure in Drosophila is a model system for studying cell sheet forces during morphogenesis. We demonstrate a role for mechanically gated ion channels (MGCs) in dorsal closure. Microinjection of GsMTx4 or GdCl₃, inhibitors of MGCs, blocks closure in a dose-dependent manner. UV-mediated uncaging of intracellular Ca2+ causes cell contraction whereas the reduction of extra- and intracellular Ca2+ slows closure. Pharmacologically blocking MGCs leads to defects in force generation via failure of actomyosin structures during closure, and impairs the ability of tissues to regulate forces in response to laser microsurgery.

We identify three genes which encode candidate MGC subunits that play a role in dorsal closure, ripped pocket, dtrpA1, and nompC. We find that knockdown of these channels either singly or in combination leads to defects in force generation and cell shapes during closure.

Our results reveal a key role for MGCs in closure, and suggest a mechanism for the coordination of force producing cell behaviors across the embryo.

Action potential firing in various excitable cells (cardiomyocytes, neurons, smooth and skeletal muscle cells) is enabled by the presence of inwardly rectifying potassium channels, such as Kir2.1, which set the resting membrane potential to a negative value allowing the activation of depolarizing voltage-gated currents. In the heart, Kir2.1-mediated IK1 current is upregulated during development, but downregulated in certain pathologies, including myocardial infarction, heart failure, and the Anderson-Tawil syndrome, a congenital disease characterized by periodic paralysis and polymorphic tachycardias. Such electrophysiological changes in the heart are often associated with profound alterations in cardiomyocyte size and shape, extracellular matrix (ECM), as well as cytoskeletal tension. Cardiomyocytes sense physical changes in their environment through integrins, heterodimeric receptors for ECM proteins that relay information to focal adhesions (FAs), force-sensitive sub-cellular structures connected to actin cytoskeleton. In different systems, dynamic changes in FA proteins as well as current flow through ion channels, including Kir2.1, are known to govern important cellular processes, including survival, proliferation, differentiation, migration, and matrix remodeling. Still, relationships between ion channels, FAs, and mechanosensitive cell electrophysiology are not well-understood. The objective of this thesis has been to explore how changes in integrin engagement and FA assembly within excitable cells affect Kir2.1 membrane localization, IK1 amplitude, and action potential characteristics. We hypothesized that integrin engagement will increase the number of active Kir2.1 channels to the membrane, thus increasing IK1 amplitude and changing action potential characteristics.

To accomplish this objective, we utilized a monoclonal line of HEK293 cells engineered to express fluorescently tagged Kir2.1 to visualize channels, a monoclonal line of HEK293 cells (“Ex293”) engineered to express Kir2.1, cardiac sodium channel Nav1.5, and gap junctional channel Connexin-43 as a well-defined excitable cell source, and neonatal rat cardiomyocytes as native excitable cells. Using microfabrication techniques, we created a platform for robustly and precisely controlling cell shape and size. Combining this technique with single cell electrophysiology and quantitative image analysis, we characterized local and global membrane distributions of Kir2.1, IK1 amplitude, and FAs. We established that membrane-bound Kir2.1 localizes in proximity to FAs, giving a non-uniform distribution of IK1 in the cell membrane.

Next we applied micropatterning of ECM proteins, pharmacological, and environmental manipulations to alter FA size and distribution in individual cells. Combining these techniques with single cell electrophysiology, confocal and total internal reflection fluorescence (TIRF) microscopy, and quantitative image analysis, we provide evidence that FAs and active integrins play a critical role in regulating Kir2.1 membrane localization, IK1 amplitude, and action potential morphology in excitable cells.

Furthermore, by studying Kir2.1 turnover dynamics using fluorescence recovery after photobleaching (FRAP), we show that the channels are uniformly transported to the membrane, where they preferentially accumulate near FA-rich sites via the local inhibition of dynamin-mediated endocytosis. We conclude this thesis with a modeling summary of the Kir2.1 trafficking and localization near FAs at the membrane.

Overall, this thesis shows links between the electrophysiology of ion channels and FA biology, coupling action potential dynamics to changes in the cellular environment and cytoskeletal mechanics. Our results propose a novel mechanism whereby engaged integrins are an important regulator of the membrane localization of Kir2.1 channels via the local inhibition of dynamin-dependent endocytosis. This mechanism renders the IK1 and cellular electrophysiology indirectly mechanosensitive to various intra- and extracellular signals affecting integrin engagement and FA dynamics. The work in this thesis warrants future in-depth studies of how cell-matrix interactions modulate the function of various ion channels across diverse cell types and pathophysiological conditions.