Biology subjects

Pushkarsky, I.

Publications and source records attributed to Pushkarsky, I..

3 recordsLinked to original sources

bioRxiv2026

Dynamic regulation of single-cell contractile heterogeneity in the human trabecular meshwork

Altered trabecular meshwork (TM) mechanics contribute to increased aqueous humor outflow resistance and elevated intraocular pressure in glaucoma, yet how contractility is regulated across individual TM cells remains poorly understood. Using a high-throughput single-cell contractility assay, we quantified time-resolved contractile behavior in primary human TM cells under defined extracellular matrix (ECM) compositions, substrate stiffnesses, and pharmacologic perturbations, and related functional mechanical behavior to intracellular protein abundance. Single-cell analyses revealed that TM contractility is not a uniform population property but a context-dependent contractile state shaped by both extracellular and intracellular regulation. ECM composition established distinct contractile programs, with collagen IV promoting rapid convergence toward a highly contractile state, collagen I supporting a slower but similar response, and fibronectin maintaining a persistently heterogeneous low-contractility state. In contrast, the intracellular perturbation dexamethasone (DEX) did not uniformly increase contractility but selectively prolonged contractile activity within the most mechanically active cells, thereby sustaining highly contractile states. This response was progressively constrained as ECM stiffness increased and was absent on pathologically stiff substrates, demonstrating that extracellular mechanical context conditions glucocorticoid responsiveness. Conversely, ROCK inhibition overrode both basal and DEX-induced contractile programs, collapsing the heterogeneous population into a uniformly low-contractility state. Finally, intracellular protein abundance poorly predicted functional mechanical behavior, with weak pooled associations reflecting divergent donor-specific relationships rather than consistent molecular-mechanical coupling. Together, these findings show that TM contractility emerges from integrating intrinsic cellular state and the extracellular mechanical environment rather than from either alone. More broadly, they identify single-cell contractile state as a functional dimension of TM cell identity that cannot be inferred from static molecular marker abundance or population-averaged mechanical measurements.

cell biology↗

bioRxiv2025

Building an atlas of mechanobiology: high-throughput contractility screen of 2418 kinase inhibitors in five primary human cell types reveals selective divergent responses among related cell types

Cellular mechanical forces play crucial roles in both normal physiology and disease, yet drug discovery efforts targeting mechanobiology have been limited in part by assumptions about the conservation of contractile pathways across cell types. Here, we present the first high-throughput contractility screen of an annotated kinase inhibitor library, evaluating 2,418 compounds across five primary human cell types using the FLECS (Fluorescent Elastomer Contractility Sensors) platform. Quantification of contractile responses revealed selective divergent responses among related cell types. Clustering analysis identified distinct mechanobiological profiles and novel pathway associations that challenge the assumption that contractile pathways are too highly conserved for selective targeting. This systematic approach supports wider adoption of mechanical phenotypic screening as a viable strategy for discovering cell-type specific contractile pathway modulators for a broad range of mechanically-driven disease indications.

bioengineering↗

bioRxiv2023

FLECS Technology for High-Throughput Screening of Hypercontractile Cellular Phenotypes in Fibrosis: A Function-First Approach to Anti-Fibrotic Drug Discovery

The pivotal role of myofibroblast contractility in the pathophysiology of fibrosis is widely recognized, yet HTS approaches are not available to quantify this critically important function in drug discovery. We develop, validate, and scale-up a HTS platform that quantifies contractile function of primary human lung myofibroblasts upon treatment with pro-fibrotic TGF-{beta}1. With the fully automated assay we screened a library of 40,000 novel small molecules in under 80 h of total assay run-time. We identified 42 hit compounds that inhibited the TGF-{beta}1-induced contractile phenotype of myofibroblasts, and enriched for 19 that specifically target myofibroblasts but not phenotypically related smooth muscle cells. Selected hits were validated in an ex vivo lung tissue models for their inhibitory effects on fibrotic gene upregulation by TGF-{beta}1. Our results demonstrate that integrating a functional contraction test into the drug screening process is key to identify compounds with targeted and diverse activity as potential anti-fibrotic agents.

bioengineering↗