3D Spatial Interactomics Maps the Dynamics of NF-κB Multiprotein Signalosomes in Single Cells

A 3D spatial interactomics workflow was established that uses iseqPLA to detect endogenous NF-κB protein-protein proximity events, representing each event by a rolling-circle amplification product and treating clusters of co-localized puncta as a measure of supercomplex spatial organization. The text states that how NF-κB signalosomes are organized in space and time within the 3D interior of a single cell had remained uncharacterized; this work couples proximity ligation readout with spinning disk confocal microscopy and 3D reconstruction to place interaction measurements in a 3D volume. The methodological description is explicit: iseqPLA, spinning disk confocal microscopy, and 3D reconstruction, applied across multiple cell systems (NIH-3T3, CF patient-derived macrophage co-cultures with IMR-90, and an independent set of healthy- and CF-donor monocyte-fibroblast co-cultures); the loaded text is summary-level and does not provide sample sizes or statistics.

3D volumetric quantification reduces the variance in nuclear-to-cytoplasmic ratio measurements relative to 2D projections. This is one of the three findings listed in the text, directly comparing 3D volumetric quantification with 2D projections for nuclear-to-cytoplasmic ratio measurement. The text phrases this as 'reduces the variance,' a quantitative methodological comparison; no variance values, sample sizes, or statistical test details are given in the text.

The choice of extracellular matrix coating shapes the fraction of NF-κB-responsive cells. The text presents matrix coating as a factor influencing the fraction of NF-κB-responsive cells, suggesting that microenvironmental conditions contribute to response heterogeneity. The text phrases this as 'shapes the fraction of NF-κB-responsive cells,' an association observed in the cell systems used; no specific fraction values or dose-response relationships are reported in the text.

In a CF model, CF airway-conditioned macrophages amplify paracrine NF-κB signaling in neighboring fibroblasts. The text lists this as one of the three findings, linking CF-derived macrophages to paracrine signal amplification in neighboring fibroblasts. This conclusion rests on CF patient-derived macrophage co-cultures with IMR-90 human fibroblasts and on an independent set of healthy- and CF-donor monocyte-fibroblast co-cultures profiled by 3D iseqPLA; the text does not report effect sizes or statistical details.

This work is aimed at researchers who need to track protein complex assembly and dissociation in single-cell 3D space, and it applies to experimental settings that read out protein-protein proximity events by proximity ligation and characterize supercomplexes through co-localized puncta clusters; the results described come from NIH-3T3 mouse fibroblasts, CF patient-derived macrophage co-cultures with IMR-90 human fibroblasts, and an independent set of healthy- and CF-donor monocyte-fibroblast co-cultures, covering supercomplex dissociation, p65 nuclear translocation, and negative-feedback engagement across cytokine time courses. Its transferability lies in bringing 3D volumetric quantification, matrix coating conditions, and paracrine signal amplification into one analytical frame for continued use in inflammation-related cells and disease models.

The loaded text is summary-level and contains no figures, sample sizes, statistics, or effect sizes, so the magnitude of the 3D-versus-2D variance comparison, the specific values by which matrix coating shapes the responsive-cell fraction, and the quantitative extent of paracrine amplification by CF airway-conditioned macrophages cannot be judged from the text; how the scGPT model fine-tuned on curated transcriptomic datasets places the NF-κB gene panel within an inflammation-relevant feature space, including its specific features and validation, would require the original figures; and the reproducibility of these findings in other cell types, other species, or in vivo settings remains an open question.