Biology subjects

Rouzbeh, N.

Publications and source records attributed to Rouzbeh, N..

4 recordsLinked to original sources

bioRxiv2026

Scalable longitudinal imaging and transcriptomics of cells in dynamic enclosures

Dynamic transitions between cell states underlie both normal physiology and disease. However, most single-cell technologies capture only static snapshots. To address this gap, we developed a platform that integrates light-guided hydrogel polymerization with computer vision to generate on-demand compartments around live cells, enabling longitudinal imaging of cellular behavior paired with whole-transcriptome profiling of the same cells at scale. These data link dynamic phenotypes with molecular programs, enabling deeper characterization of cellular states. This approach revealed an adaptive, drug-resistant state in lung cancer cells characterized by potassium channel upregulation and p53-dependent quiescence. In models of adipogenesis and microglial phagocytosis, joint analysis of imaging and transcriptomic data identified key drivers of cellular function that were missed by transcriptomic clustering alone. These results establish the value of paired functional and transcriptomic analysis to resolve molecular drivers of complex cellular behaviors.

systems biology↗

bioRxiv2026

Linking live-cell behavior to transcriptional responses across perturbations using dynamic caging

Single-cell technologies, encompassing molecular, morphological, and functional assays, have emerged as cornerstones of modern biological research and discovery. However, current experimental methods often fail to explicitly link these omic modalities, especially in live cells or longitudinally through time, impeding the study of multi-scale interactions and mechanisms of regulation. CellCage Enclosure (CCE) technology overcomes these limitations by dynamically compartmentalizing cells, allowing for scalable, live-cell, longitudinal exploration and simultaneous analysis of transcriptomic, proteomic, and morphological profiles. Using this novel technology, we generate previously inaccessible insights across various in vitro cellular systems under a diverse set of perturbations, including the discovery of morphological and proteomic features linked to immune suppressive gene set expression in human primary regulatory T cells (Tregs), as well as direct association of morphological and proteomic features with inflammatory gene modules in human colonic fibroblasts. We then develop a novel pooled CRISPR genetic screening technology using CCEs, PERTURB-LINK (PERTURBational LINKage of transcriptomics and imaging in single cells via enclosure-based screening) and apply this approach in murine bone marrow derived macrophages (BMDMs), enabling multiomic dissection of NF-{kappa}B pathway regulation in response to lipopolysaccharide (LPS) stimulation. Together, these findings demonstrate the broad impact that advancements in live-cell, paired multimodal technologies, especially upon perturbation, may offer in deepening our understanding of cellular biology.

cell biology↗

bioRxiv2026

Native architecture, allosteric modulation and gating mechanism of glycine-dependent NMDA receptors

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory signaling essential for synaptic plasticity and memory. Unlike GluN2-containing NMDARs, GluN3-containing receptors are activated solely by glycine, exhibit profound desensitization and paradoxical potentiation by GluN1-selective antagonists, including CGP-78608 (CGP). Although GluN3 NMDARs regulate synapse pruning, excitotoxicity, and are associated with schizophrenia, autism and stroke, their native stoichiometry and gating mechanism poorly defined. Using single-molecule pulldown analysis, we show that native GluN3A receptors are diheteromeric assemblies. Cryo-EM analysis of GluN1/GluN3A receptors in antagonist-bound, pre-active, active, and desensitized states, augmented by electrophysiology and pharmacology experiments, show how glycine activates the receptor solely via GluN3A-dependent conformational changes, opening the gate with [~]2-fold symmetry, and induces a [~]4-fold symmetric desensitized state. CGP-bound GluN1 restricts GluN3A rotation, promoting glycine-induced activation by blocking desensitization. These findings illuminate how CGP potentiates GluN3A receptor activity, place the receptor gating mechanism on a solid structural foundation, and define the molecular basis for pharmacological modulation.

neuroscience↗

bioRxiv2025

UCM-A86 is a selective positive allosteric modulator of GluN1/GluN3 NMDA receptors

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission in the central nervous system (CNS) where they play critical roles in normal and pathological brain functions and neurodevelopment. While the glutamate/glycine-activated GluN2-containing NMDA receptors (GluN1/GluN2) have been extensively studied, the physiological roles and pharmacology of glycine-activated GluN3-containing receptors (GluN1/GluN3) remain less understood. Although GluN1/GluN3 receptors exhibit unique functional properties and play distinct roles in neuronal development and synapse maturation, studies of their precise roles in neurophysiology and circuit function are impeded by limited availability of GluN3-selective pharmacological tools. This study describes UCM-A86, a novel GluN3-selective positive allosteric modulator, with EC50 values of 21 {micro}M and 19 {micro}M at GluN1/GluN3A and GluN1/GluN3B receptors, respectively. UCM-A86 selectively potentiates recombinant GluN1/GluN3A and GluN1/GluN3B receptors by 436% and 174%, respectively, relative to activation by glycine, with no activity at recombinant GluN1/GluN2A-D receptors. Furthermore, UCM-A86 selectively potentiates responses from native GluN1/GluN3A receptors expressed in somatostatin-expressing interneurons of the somatosensory cortex with no modulation of hippocampal AMPA receptor- and GluN1/2 NMDA receptor-mediated excitatory postsynaptic currents. Mechanistic studies suggest that UCM-A86 modulation is facilitated by agonist binding (or channel gating) and that UCM-A86 primarily potentiates GluN1/GluN3A by increasing open probability with no effects on mean channel conductance. These findings advance the synthetic pharmacology of GluN1/GluN3 receptors and provide a novel tool for modulation of native GluN3-containing NMDA receptors. Significance statementThis study introduces UCM-A86 as the first positive allosteric modulator selective for GluN3-containing NMDA receptors, addressing a critical gap in the pharmacological toolbox for investigating these understudied receptor subtypes. Using electrophysiological approaches in both recombinant and native systems, UCM-A86 demonstrates specific modulation of GluN3-containing NMDA receptors without affecting GluN2-containing NMDA receptors or AMPA receptors. UCM-A86 therefore provides new avenues to investigate the physiological roles of GluN3 subunits in normal and pathological brain function.

neuroscience↗