Biology subjects

Hoffman, B. D.

Publications and source records attributed to Hoffman, B. D..

10 recordsLinked to original sources

bioRxiv2026

Talin controls the spatial distribution of vinculin tension in focal adhesions

Cells transmit force between the extracellular matrix and the actin cytoskeleton through integrin adhesion complexes centred on talin and vinculin. Vinculin binds talin through -helical vinculin-binding sites (VBS) that are exposed when talin rod domains unfold under force. Dissecting the significance of this interaction has relied heavily on the A50I mutation in vinculin, which has been widely used as a talin-binding-null mutant. Here we show that although the A50I mutation abolishes binding to the -catenin VBS, it retains nanomolar-affinity binding to multiple talin VBS. We therefore designed an improved mutant, I12K/A50I, that eliminates this residual talin binding. Biochemical assays and single-molecule stretching experiments demonstrate that I12K/A50I VD1 fails to bind talin even when VBS are exposed by force. Using vinculin tension and conformation sensors, we show that talin binding is required for efficient recruitment of vinculin to focal adhesions and for establishing spatial gradients of vinculin tension. However, vinculin can still experience mechanical load in the absence of talin binding. These results demonstrate that A50I is not a talin-binding-null and reveal that, while talin is not required for vinculin loading, it is essential for organising the spatial distribution of mechanical load within adhesion complexes.

cell biology↗

bioRxiv2026

Physical continuity at biomaterial-ECM interfaces regulates fibroblast activation via NF-κB

Fibrotic responses at biomaterial-tissue interfaces limit implant integration and regenerative healing, yet how the interaction between biomaterials and the extracellular matrix (ECM) regulates fibroblast activation remains poorly understood. Granular hydrogels including microporous annealed particle scaffolds (MAP) reduce fibrosis, while chemically and mechanically matched hydrogels do not, suggesting a dominant role for scaffold architecture. In this model, MAP scaffolds allow collagen infiltration and form physically continuous composites, whereas hydrogels exclude collagen and generate interfacial slip planes. To isolate how biomaterial architecture influences extracellular matrix (ECM) integration and fibroblast activation, we developed a reductionist in vitro model that integrates collagen type I with either microporous annealed particle (MAP) scaffolds or chemically and mechanically matched bulk hydrogels. This physical integration stabilizes collagen architecture, limits fibroblast-mediated matrix compaction, suppresses contractility, and attenuates myofibroblast transition. Fibroblasts in mechanically integrated environments exhibit reduced expression and nuclear localization of NF-{kappa}B and are enriched for quiescent phenotypes. Together, these findings identify biomaterial-ECM physical continuity as a design principle for limiting fibrotic signaling.

bioengineering↗

bioRxiv2025

On-demand delivery of fibulin-1 enables repeated basement membrane stretching

Basement membrane (BM) extracellular matrices enwrap and structurally support tissues. Whether BMs are uniquely constructed to support tissues that undergo repetitive stretching and recoil events is unknown. During C. elegans ovulation, the spermathecal BM stretches [~]1.7-fold and then returns to its original shape every twenty minutes to passage hundreds of oocytes. Through live fluorescence microscopy, we discovered that ovulating oocytes secrete and deliver the fibulin-1 extracellular matrix protein to the spermathecal BM during stretching, where it forms a dynamic overlapping network with type IV collagen. Fibulin-1 depletion led to a breakdown in type IV collagen and BM organization, resulting in a more deformable BM and extended spermatheca. Moreover, perturbation to fibulin-1 network formation via mutagenesis was sufficient to disrupt organ shape. Together, our study reveals an on-demand fibulin-1 delivery system that protects the BM network when it is stretched, thereby allowing repeated rounds of organ expansion and recovery.

cell biology↗

bioRxiv2024

Vinculin Y822 phosphorylation regulates cardiomyocyte adhesion dynamics and adherens junction maturation in the heart

In the heart, cell-matrix and cell-cell adhesions reorganize in response to increased cardiac demand and growth to promote cardiomyocyte maturation. Vinculin, a mechanosensitive adaptor protein, links filamentous actin to cell-matrix and cell-cell adhesions and is thus positioned to regulate adhesion reorganization. However, how the two adhesion systems are coordinated in the heart, and the role of vinculin in this process is poorly understood. Here, we define the role of vinculin phosphorylation at tyrosine residue 822 (pY822) in cardiomyocyte adhesion and heart function. We found that pY822 correlated with dynamic junction remodeling in the developing heart but was lost as junctions matured postnatally. We then mutated Y822 to phenylalanine (Y822F) in the mouse to determine pY822 function in vivo. Homozygous mutant Vcl Y822F mice were viable and exhibited normal cardiac function at ten weeks of age; however, cardiac dysfunction was observed at 28 weeks. Vinculin and adherens junction proteins were reduced at cardiomyocyte junctions in Y822F hearts. In contrast, 5/{beta}1 integrin and fibronectin increased along the lateral border of Y822F cardiomyocytes. Our results demonstrate that vinculin Y822 phosphorylation regulates the balance between cadherin and integrin adhesion organization, highlighting the importance of post-translational modification in modulating vinculin function in heart physiology.

cell biology↗

bioRxiv2024

Detection of Fluorescent Protein Mechanical Switching in Cellulo

ABSTRACT/SUMMARYThe ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Forster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. MOTIVATIONThe ability of cells to sense mechanical forces is critical in developmental, physiological, and pathological processes. Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to directly probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo. This enables the visualization of force-sensitive protein function inside living cells.

cell biology↗

bioRxiv2024

Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness

Epigenetic control of cellular transcription and phenotype is influenced by changes in the cellular microenvironment, yet how mechanical cues from these microenvironments precisely influence epigenetic state to regulate transcription remains largely unmapped. Here, we combine genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA-seq CRISPR screening to identify a new class of genomic enhancers that responds to the mechanical microenvironment. These mechanoenhancers could be active on either soft or stiff extracellular matrix contexts, and regulated transcription to influence critical cell functions including apoptosis, mechanotransduction, proliferation, and migration. Epigenetic editing of mechanoenhancers on rigid materials tuned gene expression to levels observed on softer materials, thereby reprogramming the cellular response to the mechanical microenvironment. These editing approaches may enable the precise alteration of mechanically-driven disease states.

genomics↗

bioRxiv2023

Analysis of intracellular transport dynamics using quantitative phase imaging and FRET-based calcium sensors

Understanding cellular responses to mechanical environmental stimuli is an essential goal of cellular mechanotransduction studies and remains a target for microscopy technique development. While fluorescence microscopy has been advanced for this purpose - its molecular sensitivity, the ability of quantitative phase imaging to visualize subcellular structure has yet to be widely applied, perhaps due to its limited specificity. Here we seek to combine Quantitative Phase Imaging (QPI) with a molecularly sensitive Forster resonance energy transfer (FRET) construct for cell mechanotransduction studies. The multimodal imaging instrument is applied to examine cellular response to hypo-osmotic stimulus by observing the influx of calcium ions using a FRET based sensor coupled with mapping of the redistribution of intracellular mass using QPI. The combined imaging modality enables discrimination of cell response by localized region and reveals distinct behavior for each. The analysis shows cell flattening and oscillatory mass transport in response to the stimulus. With the broad array of FRET sensors under development, the combination with QPI offers new avenues for studying cell response to environmental stimuli.

biophysics↗

bioRxiv2023

Coupling During Collective Cell Migration is Controlled by a Vinculin Mechanochemical Switch

Collective cell migration (CCM) plays important roles in development, physiological, and pathological processes. A key feature of CCM is the dynamic mechanical coupling between cells, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to adapt to forces. Recent efforts have identified key proteins and implicated cellular-scale mechanical properties, but how key proteins give rise to these larger-scale mechanical processes is unclear. Using force-sensitive biosensors, cell migration assays, and molecular clutch models, we sought a molecular understanding of adhesion strengthening that could bridge this gap. We found that the mechanical linker protein vinculin bears substantial loads at AJs, FAs, and in the cytoplasm during epithelial sheet migration, and we identified a switch-like residue on vinculin that regulates its conformation and loading at the AJs during CCM. In vinculin KO-rescue, this switch jointly controlled the speed and coupling length-scale of CCM, which suggested changes in adhesion-based friction. To test this, we developed molecularly detailed friction clutch models of the FA and AJ. They show that open, loaded vinculin increases friction in adhesive structures, with larger affects observed in AJs. Thus, this work elucidates how load-bearing linker proteins can be regulated to alter mechanical properties of cells and enable rapid tuning of mechanical coupling in CCM.

cell biology↗

bioRxiv2020

Mechanically Induced Integrin Ligation Mediates Intracellular Calcium Signaling with Single Pulsating Cavitation Bubbles

Ultrasound or shockwave-induced cavitation is used therapeutically to stimulate neural and muscle tissue, but the mechanisms underlying this mechanotransduction are unclear. Intracellular Ca2+ signaling is one of the earliest events in mechanotransduction. In this study, we investigate the mechanism of Ca2+ signaling in individual HEK293T cells stimulated by single cavitation bubbles. Ca2+ responses are rare at cell-bubble distance that avoids membrane poration, even with overexpression of the mechanosensitive ion channel Piezo1, but could be increased in frequency to 42% of cells by attaching RGD beads to the apical surface of the cells. By using Piezo1 knockout and Piezo1-expressing cells, integrin-blocking antibodies, and inhibitors of P2X ion channels, key molecular players are identified in the RGD bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggers ATP release and activation of P2X--but not Piezo1--ion channels. These molecular players have not been examined previously in cavitation-induced Ca2+ signaling. The resultant Ca2+ influx causes dynamic changes in cell spread area. This approach to eliciting a Ca2+ response with cavitation microbubbles without cell injury, and the uncovered mechanotransduction mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

biophysics↗

bioRxiv2018

Tunable molecular tension sensors reveal extension-based control of vinculin loading

Molecular tension sensors have contributed to a growing understanding of mechanobiology. However, the limited dynamic range and inability to specify the mechanical sensitivity of these sensors has hindered their widespread use in diverse contexts. Here, we systematically examine the components of tension sensors that can be altered to improve their functionality. Guided by the development of a first principles model describing the mechanical behavior of these sensors, we create a collection of sensors that exhibit predictable sensitivities and significantly improved performance in cellulo. Utilized in the context of vinculin mechanobiology, a trio of these new biosensors with distinct force- and extension- sensitivities reveal that an extension-based control paradigm regulates vinculin loading. To enable the rational design of molecular tension sensors appropriate for diverse applications, we predict the mechanical behavior, in terms of force and extension, of additional 1020 distinct designs.

biophysics↗