Mechanical integration of actin and adhesion dynamics in cell migration.

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a rare autosomal codominant disease caused by mutations within the SERPINA1 gene. The most prevalent variant in patients is PiZ SERPINA1, containing a single G > A transition mutation. PiZ alpha-1 antitrypsin (AAT) is prone to misfolding, leading to the accumulation of toxic aggregates within hepatocytes. In addition, the abnormally low level of AAT secreted into circulation provides insufficient inhibition of neutrophil elastase within the lungs, eventually causing emphysema. Cytosine and adenine base editors enable the programmable conversion of C⋅G to T⋅A and A⋅T to G⋅C base pairs, respectively. In this study, two different base editing approaches were developed: use of a cytosine base editor to install a compensatory mutation (p.Met374Ile) and use of an adenine base editor to mediate the correction of the pathogenic PiZ mutation. After treatment with lipid nanoparticles formulated with base editing reagents, PiZ-transgenic mice exhibited durable editing of SERPINA1 in the liver, increased serum AAT, and improved liver histology. These results indicate that base editing has the potential to address both lung and liver disease in AATD.

Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes.

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.

Cooperative coupling of cell-matrix and cell-cell adhesions in cardiac muscle.

Adhesion between cardiac myocytes is essential for the heart to function as an electromechanical syncytium. Although cell-matrix and cell-cell adhesions reorganize during development and disease, the hierarchical cooperation between these subcellular structures is poorly understood. We reasoned that, during cardiac development, focal adhesions mechanically stabilize cells and tissues during myofibrillogenesis and intercalated disc assembly. As the intercalated disc matures, we postulated that focal adhesions disassemble as systolic stresses are transmitted intercellularly. Finally, we hypothesized that pathological remodeling of cardiac microenvironments induces excessive mechanical loading of intercalated discs, leading to assembly of stabilizing focal adhesions adjacent to the junction. To test our model, we engineered µtissues composed of two ventricular myocytes on deformable substrates of tunable elasticity to measure the dynamic organization and functional remodeling of myofibrils, focal adhesions, and intercalated discs as cooperative ensembles. Maturing µtissues increased systolic force while simultaneously developing into an electromechanical syncytium by disassembling focal adhesions at the cell-cell interface and forming mature intercalated discs that transmitted the systolic load. We found that engineering the microenvironment to mimic fibrosis resulted in focal adhesion formation adjacent to the cell-cell interface, suggesting that the intercalated disc required mechanical reinforcement. In these pathological microenvironments, µtissues exhibited further evidence of maladaptive remodeling, including lower work efficiency, longer contraction cycle duration, and weakened relationships between cytoskeletal organization and force generation. These results suggest that the cooperative balance between cell-matrix and cell-cell adhesions in the heart is guided by an architectural and functional hierarchy established during development and disrupted during disease.

Spatiotemporal constraints on the force-dependent growth of focal adhesions.

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.

Advances in high-resolution MS and hepatocyte models solve a long-standing metabolism challenge: the loratadine story.

BACKGROUND: Loratadine (LOR, Claritin(®)) is a long-acting antihistamine used to treat allergic rhinitis. The major active human metabolite, desloratadine (DL, Clarinex(®)), is extensively metabolized to 3-hydroxydesloratadine (3-OH-DL) (M40) and subsequently glucuronidated before elimination. This study revealed the ability of a novel, long-term hepatocyte micropatterned co-culture (MPCC) model to generate in vivo metabolites. Metabolites were detected and characterized using non-targeted MS/MS(ALL) with SWATH™ acquisition by a UHPLC-Q-TOF system. Results & methodology: Human MPCCs extensively metabolized LOR and formed 3-OH-DL-glucuronide (M13). Cross-species comparisons revealed monkey- and rat-specific metabolites with gender-specific DL-pyridine-N-oxide formation in male rats. These results demonstrate a first for an in vitro hepatocyte model to generate circulating metabolites of LOR and detect species-specific differences. Early focus on human metabolites could have spared characterization of nonhuman metabolites in preclinical species.

Coupling primary and stem cell-derived cardiomyocytes in an in vitro model of cardiac cell therapy.

The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (µtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell-derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell-cell junction that degrade force transmission between cells. Moreover, we developed a computational model of µtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell-cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes.

Structural phenotyping of stem cell-derived cardiomyocytes.

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes.

Quality metrics for stem cell-derived cardiac myocytes.

Advances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays.

The contractile strength of vascular smooth muscle myocytes is shape dependent.

Vascular smooth muscle cells in muscular arteries are more elongated than those in elastic arteries. Previously, we reported changes in the contractility of engineered vascular smooth muscle tissue that appeared to be correlated with the shape of the constituent cells, supporting the commonly held belief that elongated muscle geometry may allow for the better contractile tone modulation required in response to changes in blood flow and pressure. To test this hypothesis more rigorously, we developed an in vitro model by engineering human vascular smooth muscle cells to take on the same shapes as those seen in elastic and muscular arteries and measured their contraction during stimulation with endothelin-1. We found that in the engineered cells, actin alignment and nuclear eccentricity increased as the shape of the cell elongated. Smooth muscle cells with elongated shapes exhibited lower contractile strength but greater percentage increase in contraction after endothelin-1 stimulation. We analysed the relationship between smooth muscle contractility and subcellular architecture and found that changes in contractility were correlated with actin alignment and nuclear shape. These results suggest that elongated smooth muscle cells facilitate muscular artery tone modulation by increasing its dynamic contractile range.

Dynamic and structural signatures of lamellar actomyosin force generation.

The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ∼60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.

Transient frictional slip between integrin and the ECM in focal adhesions under myosin II tension.

BACKGROUND: The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. Although adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown. RESULTS: To elucidate the nature of force transmission as myosin II tension is applied to focal adhesions, we correlated the dynamics of focal adhesion proteins and the actin cytoskeleton to local traction stresses. Under low extracellular tension, newly formed adhesions near the cell periphery underwent a transient retrograde displacement preceding elongation. We found that myosin II-generated tension drives this mobility, and we determine the interface of differential motion, or "slip," to be between integrin and the ECM. The magnitude and duration of both adhesion slip and associated F-actin dynamics is strongly modulated by ECM compliance. Traction forces are generated throughout the slip period, and adhesion immobilization occurs at a constant tension. CONCLUSIONS: We have identified a tension-dependent, extracellular "clutch" between integrins and the extracellular matrix; this clutch stabilizes adhesions under myosin II driven tension. The current work elucidates a mechanism by which force transmission is modulated during focal adhesion maturation.

Conserved F-actin dynamics and force transmission at cell adhesions.

Adhesions are a central mechanism by which cells mechanically interact with the surrounding extracellular matrix (ECM) and neighboring cells. In both cell-ECM and cell-cell adhesions, forces generated within the actin cytoskeleton are transmitted to the surrounding environment and are essential for numerous morphogenic processes. Despite differences in many molecular components that regulate cell-cell and cell-ECM adhesions, the roles of F-actin dynamics and mechanical forces in adhesion regulation are surprisingly similar. Moreover, force transmission at adhesions occurs concomitantly with dynamic F-actin; proteins comprising the adhesion of F-actin to the plasma membrane must accommodate this movement while still facilitating force transmission. Thus, despite different molecular architectures, integrin and cadherin-mediated adhesions operate with common biophysical characteristics to transmit and respond to mechanical forces in multicellular tissue.

Preparation of complaint matrices for quantifying cellular contraction.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties. To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces. Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.