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-antigendensity tumors while maintaining their unique capacity for persistence.

In developing tissues, cells estimate their spatial position by sensing graded concentrations of diffusible signaling proteins called morphogens. Morphogen-sensing pathways exhibit diverse molecular architectures, whose roles in controlling patterning dynamics and precision have been unclear. In this work, combining cell-based in vitro gradient reconstitution, genetic rewiring, and mathematical modeling, we systematically analyzed the distinctive architectural features of the Sonic Hedgehog pathway. We found that the combination of double-negative regulatory logic and negative feedback through the PTCH receptor accelerates gradient formation and improves robustness to variation in the morphogen production rate compared with alternative designs. The ability to isolate morphogen patterning from concurrent developmental processes and to compare the patterning behaviors of alternative, rewired pathway architectures offers a powerful way to understand and engineer multicellular patterning.

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 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 cargos 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.

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.

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.

No abstract

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 Förster resonance energy transfer (FRET)-based measurements 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 broadly applicable tool for the measurement molecular-scale forces in biological systems.

A continuous sheet of epithelial cells surrounding a hollow opening, or lumen, defines the basic topology of numerous organs. De novo lumen formation is a central feature of embryonic development whose dysregulation leads to congenital and acquired diseases of the kidney and other organs. While prior work has described the hydrostatic pressure-driven expansion of lumens when they are large, the physical mechanisms that promote the formation and maintenance of small, nascent lumens are less explored. In particular, models that rely solely on pressure-driven expansion face a potential challenge in that the Laplace pressure, which resists lumen expansion, is predicted to scale inversely with lumen radius. We investigated the cellular and physical mechanisms responsible for stabilizing the initial stages of lumen growth using a 3D culture system in which epithelial cells spontaneously form hollow lumens. Our experiments revealed that neither the actomyosin nor microtubule cytoskeletons are required to stabilize lumen geometry, and that a positive intraluminal pressure is not necessary for lumen stability. Instead, our observations are in agreement with a quantitative model in which cells maintain lumen shape due to topological and geometrical factors tied to the establishment of apicobasolateral polarity. We suggest that this model may provide a general physical mechanism for the formation of luminal openings in a variety of physiological contexts. Lumen shapes for small-and intermediate-size MDCK spheroids are inconsistent with a Laplace model of lumen stabilityWe seeded MDCK cells expressing a fluorescent marker for actin filaments (Lifeact-RFP) in the recombinant extracellular matrix Matrigel. Under these culture conditions, MDCK cells spontaneously form hollow spheroids within 24 hours. To obtain high-resolution images of nascent lumens, we imaged young (18-24 hours) spheroids using lattice light sheet microscopy (LLSM). This acquisition method produced images in which the two opposing apical cortices of the lumen are clearly distinguishable and separated by ~200-300 nm (Fig. 1b,c). We infer that cellular apical surfaces are intrinsically non-adherent, as even small fluctuations in cell shape would allow apposing apical surfaces to contact and potentially adhere. This "non-stick" behavior may reflect the enrichment of negatively charged sialoglycoproteins such as