Alexander R. Dunn

Endothelial cells (EC) form a dynamic monolayer barrier that regulates vascular permeability and is highly sensitive to mechanical cues, including shear stresses (SS) generated by blood flow. While spatial shear stress gradients (SSG) are linked to vascular pathologies such as atherosclerosis, their direct effects on cellular biomechanics across EC subtypes remain underexplored. Here, using an impinging flow device compatible with videomicroscopy, we demonstrate that exposure of two EC types to SSG causes an immediate and sustained two-fold decrease in migration speed, accompanied by enhanced coordination of collective motion and directional migration against the flow. Concurrently, EC traction and monolayer stresses increase acutely upon flow onset and continue to rise linearly over time. Mechanistically, FAK-mediated mechanotransduction governs flow-opposed migration, whereas the time-dependent increase in traction depends on TGF-β1 signaling and actomyosin contractility. Together, these findings identify SSG as key regulators of EC mechanics, collective dynamics, and signaling, with important implications for vascular disease diagnostics and therapeutics.

Summary: Precise control of synapse formation is essential for brain function. Adhesion-GPCRs combine adhesion, signaling, and scaffold recruitment to regulate synapse formation, yet their mechanisms of action remain unclear. Here, we identify nanoscale clustering of the adhesion-GPCR Latrophilin-3 as a key mechanism regulating excitatory synapse assembly. We find that endogenous Latrophilin-3 forms densely packed nanoclusters, with larger nanoclusters enriched at excitatory synapses. To probe the functional significance of Latrophilin-3 clustering, we engineered an optogenetically controlled Latrophilin-3. Light-induced Latrophilin-3 clustering triggered robust excitatory synapse formation, which required both Latrophilin-3-mediated G-protein signaling and postsynaptic scaffold recruitment. Strikingly, clustering of a mutant Latrophilin-3 lacking known ligand-binding domains still promoted synapse formation, indicating that Latrophilin-3 ligands induce synapse assembly primarily by clustering Latrophilin-3. Our findings suggest a general paradigm by which extracellular ligand-induced clustering activates adhesion-GPCRs by generating compartmentalized signaling, which in the case of Latrophilin-3, cooperates with postsynaptic scaffold recruitment to drive synapse assembly.

Hypertrophic cardiomyopathy (HCM) is the most common monogenic inherited heart disease and is a major cause of sudden death in individuals under 35 years of age. HCM is associated with progressive tissue-level disarray and subcellular disorganization in individual cardiomyocytes. Mutations in β-cardiac myosin (MYH7), the second most common genetic cause of HCM, commonly result in changes in sarcomeric force production, but how this leads to altered cell- and tissue-level organization is unclear. Here, we use cryo-electron tomography (cryo-ET) to bridge the molecular and cellular scales by visualizing the nanoscale organization of individual myosin-containing thick filaments within sarcomeres of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Compared to isogenic wild-type controls, hiPSC-CMs expressing P710R MYH7 exhibit pronounced disruption of the hexagonal packing of thick filaments within individual sarcomeres, which would be difficult to visualize using conventional light microscopy or even room-temperature electron microscopy. We also observe ribosome infiltration into areas of sarcomeric disorder for both wild-type and P710R MYH7 hiPSC-CMs, suggesting that disordered regions may be sites of local proteostasis or remodeling. Together, these data illuminate how altered myosin activity can propagate to yield dramatic changes in sarcomeric organization in HCM.

Endothelial cells (EC) form a dynamic monolayer barrier that regulates vascular permeability and is highly sensitive to mechanical cues, including shear stress (SS) generated by blood flow. While spatial shear stress gradients (SSG) are strongly linked to vascular pathologies such as atherosclerosis, their direct effects on EC kinematics, mechanics, and signaling, and whether these responses vary across EC subtypes, remain poorly understood. Here, using an impinging flow device compatible with long-term videomicroscopy, we demonstrate that exposure of two distinct EC types to SSG leads to an immediate and sustained two-fold decrease in migration speed, accompanied by enhanced coordination of collective motion and directional migration against the flow. Concurrently, EC traction forces and monolayer stresses increase acutely upon flow onset and continue to rise linearly over time. Mechanistically, FAK-mediated mechanotransduction governs flow-opposed migration, whereas the time-dependent increase in traction depends on TGF-β1 signaling and actomyosin contractility but is not required for setting migration direction. Together, these findings identify SSG as key regulators of EC mechanics, collective dynamics, and signaling, with important implications for vascular disease diagnostics and therapeutic strategies.

Planar cell polarity (PCP) signaling orients epithelial cells along an in-plane tissue axis and is a required feature of embryonic development in bilaterians. In PCP signaling, distinct molecular subcomplexes segregate to opposite sides of each cell, where they interact across intercellular junctions to form highly polarized clusters. How signal amplification and error correction occur to achieve the observed level of asymmetry is not known. Here, we used quantitative photobleaching to quantify the number of monomers of core PCP proteins within individual clusters in living Drosophila pupal wings. These data, combined with mathematical modeling, allowed us to determine how the growth of PCP signaling complexes is coupled to robust and accurate polarization. We find that clusters become increasingly polarized and correctly oriented as they become larger. Further, mutations that inhibit cluster growth inhibit cell- and tissue-level polarization, confirming that a population of large clusters is required for successful polarization. Our results reveal how cluster assembly provides the signal amplification and error correction necessary for robust polarization at the molecular, and hence cellular and tissue, levels, a mechanism that may apply to the many signal transduction pathways that feature the assembly of large, nonstoichiometric signalosomes.

Single-molecule force spectroscopy (SMFS) is a powerful tool to measure how biomolecules respond to mechanical force, but limited sequence throughput constrains its potential. Here, we present a single-molecule, multiplexed, microfluidic force spectroscopy (SM ³ FS) assay that uses parallelized microfluidics to measure up to 80 sequence variants per experiment. Using SM ³ FS, we stretched, overstretched, or unzipped 241 different DNA structures and generated 131,847 single-molecule traces from a quarter-million observations. High sequence throughput allowed us to identify DNA structures that are kinetically stable yet mechanically fragile (F _rupture < 3 pN at 0.5 pN s ^-1 ), revealing how mechanosensitivity can arise as an intrinsic property of multivalent systems. By enabling systematic sequence-function mapping under force, SM ³ FS opens a path to high-throughput nonequilibrium studies of biomolecules.

Force generation in the heart relies on sarcomeres in which myosin II contracts antiparallel arrays of F-actin. Sarcomeres are linked at Z-discs, where the protein α-actinin-2 crosslinks antiparallel F-actin from adjoining sarcomeres and recruits signaling proteins that regulate cardiomyocyte growth. How Z-discs self-assemble and transduce mechanical signals remains unclear. Using optical trap assays, we find that the bond between α-actinin-2 and F-actin selectively strengthens under load applied toward the pointed, (–) end, the direction of myosin force production, whereas binding is dramatically attenuated when load is oriented toward the filament (+) end. Mutations that cause hypertrophic cardiomyopathy (G111V, A119T, T247M) weaken this bond and disrupt directionality, defects that are amplified when multiple α-actinin-2 work together to anchor F-actin against (–)-end directed loads. We further demonstrate that anchoring by wild-type α-actinin-2 assists cardiac myosin II in generating force against an external load, while A119T α-actinin-2 blocks myosin force production. The directional interaction between α-actinin-2 and F-actin offers a plausible mechanism by which the antiparallel organization of actin filaments at the Z-disc, and thereby sarcomeric order as a whole, can emerge spontaneously in response to myosin-generated force. Our data likewise implicate α-actinin-2 as a key mechanosensor in the cardiac sarcomere.

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high-resolution imaging of other adherent and non-adherent cell types with cryo-ET.

Rationale Endothelial dysfunction is a key feature of pulmonary arterial hypertension (PAH). We previously identified Wnt7a, a ligand of the Wnt planar cell polarity (PCP) pathway, as essential for pulmonary angiogenesis, with its loss linked to PAH. Given the importance of Wnt/PCP to lung endothelial function and angiogenesis, our goal is to elucidate how Wnt/PCP regulates angiogenic responses in pulmonary microvascular endothelial cells (PMVECs). ROR2, a tyrosine kinase receptor specific to Wnt/PCP, is crucial for cardiovascular development, but its role in PAH is unclear. We hypothesized that ROR2 supports endothelial homeostasis, and its loss would impair angiogenesis, contributing to PAH. Methods Endothelial-specific ROR2 knockout (ROR2 ECKO) and wild-type (WT) mice were studied under normoxia and chronic hypoxia using echocardiography, hemodynamics, and lung morphometry. PMVECs from healthy and PAH lungs were transfected with ROR2 siRNA/constructs for functional and molecular studies. Focal adhesion (FA) activation and force generation were assessed via FRET-based methods. Bulk and single-cell transcriptomic analyses were performed on siROR2 PMVECs and ROR2 ECKO lungs. Results ROR2 ECKO mice exhibited worsened pulmonary hypertension, right ventricular remodeling, microvascular loss, and muscularization in hypoxia. Single-cell RNA sequencing of lung endothelial cells showed dysregulation of pathways involved in barrier formation and angiogenesis. Evans blue dye extravasation confirmed reduced endothelial barrier integrity in ROR2 ECKO mice. ROR2-deficient PAH PMVECs displayed increased adhesion, permeability, and FA numbers, with reduced VE-cadherin at cell junctions. Confocal imaging revealed ROR2 localization in FAs, interacting with integrin β1 (ITGB1). FRET analysis showed that ITGB1 remained in an active, adhesion-promoting state in ROR2-deficient cells. Restoring ROR2 in PAH PMVECs normalized adhesion, barrier function, and FA abundance. Transcriptomic analysis identified Rab12 as a key mediator of ROR2-ITGB1 crosstalk, with Rab12 knockdown mimicking ROR2 deficiency in PMVECs. Conclusions ROR2 regulates pulmonary angiogenesis by maintaining endothelial barrier integrity and facilitating integrin recycling. Restoring ROR2 signaling could be a potential therapeutic approach for PAH.