3D Spatial Interactomics Maps the Dynamics of NF-κB Multiprotein Signalosomes in Single Cells
- 1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA; Interdisciplinary Bioengineerin
Abstract
NF{kappa}B 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{kappa}B 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{kappa}B-responsive cells, and CF airway-conditioned macrophages amplify paracrine NF{kappa}B 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{kappa}B 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.
Related
- Recording proteome dynamics in the late-stage embryo at hour-timescale, spatial, and single-cell resolution with MEMBRYO
- Integration of SYT1 Interactomics and Dual-Localization Proteomics Links ER-PM Contacts to Lignin Deposition
- The Chlamydia trachomatis effector CT226 directly engages LRRFIP1 to recruit a multiprotein complex that limits inclusion size and progeny production
- Single-cell and spatial characterization of the human pancreas in cystic fibrosis
- Host behavioral responses to perceived risk shape spatial disease dynamics
BibTeX
@misc{zhang2026spatial,
title = {{3D Spatial Interactomics Maps the Dynamics of NF-κB Multiprotein Signalosomes in Single Cells}},
author = {N. Zhang and C. Leese-Thompson and H. Ozuna and B. Peng and S. Sirigireddy and D. Nambiar and L. Ramanan and R. Tirouvanziam and B. Kopp and A. F. Coskun},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751198},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751198v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - 3D Spatial Interactomics Maps the Dynamics of NF-κB Multiprotein Signalosomes in Single Cells
AU - N. Zhang
AU - C. Leese-Thompson
AU - H. Ozuna
AU - B. Peng
AU - S. Sirigireddy
AU - D. Nambiar
AU - L. Ramanan
AU - R. Tirouvanziam
AU - B. Kopp
AU - A. F. Coskun
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751198
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751198v1
AB - NF{kappa}B 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{kappa}B 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{kappa}B-responsive cells, and CF airway-conditioned macrophages amplify paracrine NF{kappa}B 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{kappa}B 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.
ER -