Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

Optical control of fast and processive engineered myosins in vitro and in living cells.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

PDZ Domains from the Junctional Proteins Afadin and ZO-1 Act as Mechanosensors.

Intercellular adhesion complexes must withstand mechanical forces to maintain tissue cohesion, while also retaining the capacity for dynamic remodeling during tissue morphogenesis and repair. Most cell-cell adhesion complexes contain at least one PSD95/Dlg/ZO-1 (PDZ) domain situated between the adhesion molecule and the actin cytoskeleton. However, PDZ-mediated interactions are characteristically nonspecific, weak, and transient, with several binding partners per PDZ domain, micromolar dissociation constants, and bond lifetimes of seconds or less. Here, we demonstrate that the bonds between the PDZ domain of the cytoskeletal adaptor protein afadin and the intracellular domains of the adhesion molecules nectin-1 and JAM-A form molecular catch bonds that reinforce in response to mechanical load. In contrast, the bond between the PDZ3-SH3-GUK (PSG) domain of the cytoskeletal adaptor ZO-1 and the JAM-A intracellular domain becomes dramatically weaker in response to ∼2 pN of load, the amount generated by single molecules of the cytoskeletal motor protein myosin II. These results suggest that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes, and that mechanical load can enhance the selectivity of PDZ-peptide interactions. These results suggest that PDZ mechanosensitivity may help to generate the intricate molecular organization of cell-cell junctions and allow junctional complexes to dynamically remodel in response to mechanical load.

Piconewton forces mediate GAIN domain dissociation of the latrophilin-3 adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-Autoproteolysis Inducing (GAIN) domain, but the two resulting fragments remain associated on the cell surface. It is thought that force-mediated dissociation of the fragments exposes a peptide that activates G-protein signaling of aGPCRs, but whether GAIN domain dissociation can occur on biologically relevant timescales and at physiological forces is unknown. Here, we show using magnetic tweezers that physiological forces dramatically accelerate the dissociation of the latrophilin-3 GAIN domain. Forces in the 1-10 pN range were sufficient to dissociate the GAIN domain on a seconds-to-minutes timescale, and the GAIN domain fragments reversibly reassociated after dissociation. Thus, mechanical force may be a key driver of latrophilin signaling during synapse formation, suggesting a physiological mechanism by which aGPCRs may mediate mechanically-induced signal transduction.

Facile detection of mechanical forces across proteins in cells with STReTCh.

Numerous proteins experience and respond to mechanical forces as an integral part of their cellular functions, but measuring these forces remains a practical challenge. Here, we present a compact, 11-kDa molecular tension sensor termed STReTCh (sensing tension by reactive tag characterization). Unlike existing genetically encoded tension sensors, STReTCh does not rely on experimentally demanding measurements based on Förster resonance energy transfer and is compatible with typical fix-and-stain protocols. Using a magnetic tweezers assay, we calibrate the STReTCh module and show that it responds to physiologically relevant, piconewton forces. As proof of concept, we use an extracellular STReTCh-based sensor to visualize cell-generated forces at integrin-based adhesion complexes. In addition, we incorporate STReTCh into vinculin, a cytoskeletal adaptor protein, and show that STReTCh reports on forces transmitted between the cytoskeleton and cellular adhesion complexes. These data illustrate the utility of STReTCh as a tool for visualizing molecular-scale forces in biological systems.

Physical basis for the determination of lumen shape in a simple epithelium.

The formation of a hollow lumen in a formerly solid mass of cells is a key developmental process whose dysregulation leads to diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by experiments in the mouse blastocyst. However, lumens formed in other tissues adopt irregular shapes with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen shape in all cases. Here we use live-cell imaging to study the physical mechanism of lumen formation in Madin-Darby Canine Kidney cell spheroids, a canonical cell-culture model for lumenogenesis. We find that in this system, lumen shape reflects basic geometrical considerations tied to the establishment of apico-basal polarity. A physical model incorporating both cell geometry and intraluminal pressure can account for our observations as well as cases in which pressure plays a dominant role.

Tuning the Antigen Density Requirement for CAR T-cell Activity.

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627.