Biology subjects

Vachharajani, V. T.

Publications and source records attributed to Vachharajani, V. T..

4 recordsLinked to original sources

bioRxiv2023

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. Many 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 multiple 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. Thus, physiologically relevant forces can exert dramatic and opposite effects on the stability of two of the major linkages between cell-cell adhesion proteins and the F-actin cytoskeleton. Our data demonstrate that that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes. More broadly, our findings suggest that mechanical force may serve as a previously unsuspected regulator of the hundreds of PDZ-ligand interactions present in animal cells.

biophysics↗

bioRxiv2023

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.

biophysics↗

bioRxiv2022

STReTCh: a strategy for facile detection of mechanical forces across proteins in cells

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 Forster 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.

bioengineering↗

bioRxiv2019

A geometry-based model describes lumen stability in epithelial cells

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. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by recent experiments in the mouse blastocyst. High luminal pressure should produce lumen surfaces that bow outwards toward the surrounding cells. However, lumens formed in other embryonic tissues adopt highly irregular shapes, with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen growth in all cases. We used three-dimensional live-cell imaging to study the physical mechanism of lumen formation in Madin Darby Canine Kidney (MDCK) cell spheroids, a canonical cell-culture model for lumenogenesis. Our experiments revealed that neither lumen pressure nor the actomyosin cytoskeleton were required to maintain lumen shape or stability. Instead, we find that, in our model system, lumen shape results from simple geometrical factors tied to the establishment of apico-basal polarity. A quantitative physical model that incorporates cell geometry, cortical tension, and intraluminal pressure can account for our observations as well as cases in which pressure indeed plays a dominant role. Our results thus support a unifying physical mechanism for the formation of luminal openings in a variety of physiological contexts.

biophysics↗