{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:24Z","timestamp":1792790364675,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,17]]},"group-title":"Bioengineering","reference-count":45,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["R35GM151028"],"award-info":[{"award-number":["R35GM151028"]}]},{"name":"National Institutes of Health","award":["T32GM142616"],"award-info":[{"award-number":["T32GM142616"]}]},{"name":"U.S. National Science Foundation","award":["2338935"],"award-info":[{"award-number":["2338935"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>NF\u03baB signaling drives inflammatory responses by rapidly assembling membrane-proximal multiprotein supercomplexes, yet how these assemblies are organized in space and time within the 3D interior of a single cell has remained uncharacterized. We addressed this by profiling endogenous NF\u03baB protein-protein interactions with an intelligent sequential proximity ligation assay (iseqPLA), read out by spinning disk confocal microscopy and 3D reconstruction. Each detected protein-protein proximity event is represented by a rolling-circle amplification product, and we treat clusters of co-localized puncta as a measure of supercomplex spatial organization. Across NIH-3T3 mouse fibroblasts, cystic fibrosis (CF) patient-derived macrophage co-cultures with IMR-90 human fibroblasts, and an independent set of healthy-and CF-donor monocyte-fibroblast co-cultures profiled by 3D iseqPLA, we tracked supercomplex dissociation, p65 nuclear translocation, and negative-feedback engagement across cytokine time courses. Three findings emerge: 3D volumetric quantification reduces the variance in nuclear-to-cytoplasmic ratio measurements relative to 2D projections, the choice of extracellular matrix coating shapes the fraction of NF\u03baB-responsive cells, and CF airway-conditioned macrophages amplify paracrine NF\u03baB signaling in neighboring fibroblasts in a CF model. A single-cell Generative Pretrained Transformer (scGPT) foundation model, fine-tuned on curated transcriptomic datasets, further places our NF\u03baB gene panel within an inflammation-relevant feature space. Together, these results establish a 3D spatial interactomics workflow for dissecting supercomplex dynamics in health and disease.<\/jats:p>","DOI":"10.64898\/2026.09.14.751198","type":"posted-content","created":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T01:30:15Z","timestamp":1789695015000},"source":"Crossref","is-referenced-by-count":0,"title":["3D Spatial Interactomics Maps the Dynamics of NF-\u03baB Multiprotein Signalosomes in Single Cells"],"prefix":"10.64898","author":[{"given":"Nicholas","family":"Zhang","sequence":"first","affiliation":[{"name":"Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA"},{"name":"Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, GA, USA"},{"name":"Parker H. 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