Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo.

Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

The α-Catenin mechanosensing M region is required for cell adhesion during tissue morphogenesis.

α-Catenin couples the cadherin-catenin complex to the actin cytoskeleton. The mechanosensitive α-Catenin M region undergoes conformational changes upon application of force to recruit interaction partners. Here, we took advantage of the tension landscape in the Drosophila embryo to define three different states of α-Catenin mechanosensing in support of cell adhesion. Low-, medium-, and high-tension contacts showed a corresponding recruitment of Vinculin and Ajuba, which was dependent on the α-Catenin M region. In contrast, the Afadin homolog Canoe acts in parallel to α-Catenin at bicellular low- and medium-tension junctions but requires an interaction with α-Catenin for its tension-sensitive enrichment at high-tension tricellular junctions. Individual M region domains make complex contributions to cell adhesion through their impact on interaction partner recruitment, and redundancies with the function of Canoe. Our data argue that α-Catenin and its interaction partners are part of a cooperative and partially redundant mechanoresponsive network that supports AJs remodeling during morphogenesis.

Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex.

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

DDR1 (Discoidin Domain Receptor-1)-RhoA (Ras Homolog Family Member A) Axis Senses Matrix Stiffness to Promote Vascular Calcification.

OBJECTIVE: Vascular calcification is a pathology characterized by arterial mineralization, which is a common late-term complication of atherosclerosis that independently increases the risk of adverse cardiovascular events by fourfold. A major source of calcifying cells is transdifferentiating vascular smooth muscle cells (VSMCs). Previous studies showed that deletion of the collagen-binding receptor, DDR1 (discoidin domain receptor-1), attenuated VSMC calcification. Increased matrix stiffness drives osteogenesis, and DDR1 has been implicated in stiffness sensing in other cell types; however, the role of DDR1 as a mechanosensor in VSMCs has not been investigated. Here, we test the hypothesis that DDR1 senses increased matrix stiffness and promotes VSMC transdifferentiation and calcification. Approach and Results: Primary VSMCs isolated from Ddr1+/+ (wild-type) and Ddr1-/- (knockout) mice were studied on collagen-I-coated silicon substrates of varying stiffness, culturing in normal or calcifying medium. DDR1 expression and phosphorylation increased with increasing stiffness, as did in vitro calcification, nuclear localization of Runx2 (Runt-related transcription factor 2), and expression of other osteochondrocytic markers. By contrast, DDR1 deficient VSMCs were not responsive to stiffness and did not undergo transdifferentiation. DDR1 regulated stress fiber formation and RhoA (ras homolog family member A) activation through the RhoGEF (rho guanine nucleotide exchange factor), Vav2. Inhibition of actomyosin contractility reduced Runx2 activation and attenuated in vitro calcification in wild-type VSMCs. Finally, a novel positive feedforward loop was uncovered between DDR1 and actomyosin contractility, important in regulating DDR1 expression, clustering, and activation. CONCLUSIONS: This study provides mechanistic insights into DDR1 mechanosignaling and shows that DDR1 activity and actomyosin contractility are interdependent in mediating stiffness-dependent increases in VSMC calcification.

Actin and myosin dynamics are independent during Drosophila embryonic wound repair.

Collective cell movements play a central role in embryonic development, tissue repair, and metastatic disease. Cell movements are often coordinated by supracellular networks formed by the cytoskeletal protein actin and the molecular motor nonmuscle myosin II. During wound closure in the embryonic epidermis, the cells around the wound migrate collectively into the damaged region. In Drosophila embryos, mechanical tension stabilizes myosin at the wound edge, facilitating the assembly of a supracellular myosin cable around the wound that coordinates cell migration. Here, we show that actin is also stabilized at the wound edge. However, loss of tension or myosin activity does not affect the dynamics of actin at the wound margin. Conversely, pharmacological stabilization of actin does not affect myosin levels or dynamics around the wound. Together, our data suggest that actin and myosin are independently regulated during embryonic wound closure, thus conferring robustness to the embryonic wound healing response.

Altering integrin engagement regulates membrane localization of Kir2.1 channels.

How ion channels localize and distribute on the cell membrane remains incompletely understood. We show that interventions that vary cell adhesion proteins and cell size also affect the membrane current density of inward-rectifier K+ channels (Kir2.1; encoded by KCNJ2) and profoundly alter the action potential shape of excitable cells. By using micropatterning to manipulate the localization and size of focal adhesions (FAs) in single HEK293 cells engineered to stably express Kir2.1 channels or in neonatal rat cardiomyocytes, we establish a robust linear correlation between FA coverage and the amplitude of Kir2.1 current at both the local and whole-cell levels. Confocal microscopy showed that Kir2.1 channels accumulate in membrane proximal to FAs. Selective pharmacological inhibition of key mediators of protein trafficking and the spatially dependent alterations in the dynamics of Kir2.1 fluorescent recovery after photobleaching revealed that the Kir2.1 channels are transported to the cell membrane uniformly, but are preferentially internalized by endocytosis at sites that are distal from FAs. Based on these results, we propose adhesion-regulated membrane localization of ion channels as a fundamental mechanism of controlling cellular electrophysiology via mechanochemical signals, independent of the direct ion channel mechanogating.

Forceful closure: cytoskeletal networks in embryonic wound repair.

Embryonic tissues heal wounds rapidly and without scarring, in a process conserved across species and driven by collective cell movements. The mechanisms of coordinated cell movement during embryonic wound closure also drive tissue development and cancer metastasis; therefore, embryonic wound repair has received considerable attention as a model of collective cell migration. During wound closure, a supracellular actomyosin cable at the wound edge coordinates cells, while actin-based protrusions contribute to cell crawling and seamless wound healing. Other cytoskeletal networks are reorganized during wound repair: microtubules extend into protrusions and along cell-cell boundaries as cells stretch into damaged regions, septins accumulate at the wound margin, and intermediate filaments become polarized in the cells adjacent to the wound. Thus, diverse cytoskeletal networks work in concert to maintain tissue structure, while also driving and organizing cell movements to promote rapid repair. Understanding the signals that coordinate the dynamics of different cytoskeletal networks, and how adhesions between cells or with the extracellular matrix integrate forces across cells, will be important to elucidate the mechanisms of efficient embryonic wound healing and may have far-reaching implications for developmental and cancer cell biology.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Improving Quality, Reproducibility, and Usability of FRET-Based Tension Sensors.

Mechanobiology, the study of how mechanical forces affect cellular behavior, is an emerging field of study that has garnered broad and significant interest. Researchers are currently seeking to better understand how mechanical signals are transmitted, detected, and integrated at a subcellular level. One tool for addressing these questions is a Förster resonance energy transfer (FRET)-based tension sensor, which enables the measurement of molecular-scale forces across proteins based on changes in emitted light. However, the reliability and reproducibility of measurements made with these sensors has not been thoroughly examined. To address these concerns, we 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 used these methods, and demonstrated that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggests FRET-based tension sensors could be coupled with a variety of immuno-fluorescent labeling techniques. 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 work establishes guidelines to improve FRET-based tension sensor measurements, validate novel implementations of these sensors, and ensure that results are precise and reproducible. © 2018 International Society for Advancement of Cytometry.

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the Förster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration.

Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection.

Electrotransfection is a widely used method for delivering genes into cells with electric pulses. Although different hypotheses have been proposed, the mechanism of electrotransfection remains controversial. Previous studies have indicated that uptake and intracellular trafficking of plasmid DNA (pDNA) are mediated by endocytic pathways, but it is still unclear which pathways are directly involved in the delivery. To this end, the present study investigated the dependence of electrotransfection on macropinocytosis. Data from the study demonstrated that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles. Furthermore, electrotransfection efficiency could be decreased significantly by reducing temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

Molecular-Scale Tools for Studying Mechanotransduction.

Mechanical stimuli are known to be potent regulators of the form and function of cells and organisms. Although biological regulation has classically been understood in terms of principles from solution biochemistry, advancements in many fields have led to the development of a suite of techniques that are able to reveal the interplay between mechanical loading and changes in the biochemical properties of proteins in systems ranging from single molecules to living organisms. Here, we review these techniques and highlight the emergence of a new molecular-scale understanding of the mechanisms mediating the detection and response of cells to mechanical stimuli, a process termed mechanotransduction. Specifically, we focus on the role of subcellular adhesion structures in sensing the stiffness of the surrounding environment because this process is pertinent to applications in tissue engineering as well the onset of several mechanosensitive disease states, including cancer.

Construction, imaging, and analysis of FRET-based tension sensors in living cells.

Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors.

A micropatterning and image processing approach to simplify measurement of cellular traction forces.

Quantification of the traction forces that cells apply to their surroundings has been critical to the advancement of our understanding of cancer, development and basic cell biology. This field was made possible through the development of engineered cell culture systems that permit optical measurement of cell-mediated displacements and computational algorithms that allow conversion of these displacements into stresses and forces. Here, we present a novel advancement of traction force microscopy on polyacrylamide (PAA) gels that addresses limitations of existing technologies. Through an indirect patterning technique, we generated PAA gels with fluorescent 1 µm dot markers in a regularized array. This improves existing traction measurements since (i) multiple fields of view can be measured in one experiment without the need for cell removal; (ii) traction vectors are modeled as discrete point forces, and not as a continuous field, using an extremely simple computational algorithm that we have made available online; and (iii) the pattern transfer technique is amenable to any of the published techniques for producing patterns on glass. In the future, this technique will be used for measuring traction forces on complex patterns with multiple, spatially distinct ligands in systems for applying strain to the substrate, and in sandwich cultures that generate quasi-three-dimensional environments for cells.