(B-49) High Throughput, Translational Dynamics in Single Cell Signaling

Introduction:: Nuclear Factor kappa B (NFκB) is a transcription factor signaling pathway that is central to innate immunity in mammals. While typically responsible for proinflammatory activation, its dysregulation has been associated with cancers, arthritis, Alzheimer’s, and autoimmune disorders ^1–4. Although its proteomic activity has been well characterized, the role of the transcriptome has not been examined. The transcriptome function has been recognized beyond sole translation; the existence of non-coding and other RNAs in cancer has been acknowledged ⁵. Theoretically, although the Central Dogma of biology holds that mRNA is translated into protein, this process has yet to be directly observed in living cells. In fact, RNA protein correlations vary wildly across cells and tissues; some correlation metrics only have 40% explanatory power of their associated proteins ⁶. Therefore, investigating the translational regulation may reveal how RNAs and associated proteins interact at the subcellular level, which likely affects signaling mechanisms and disease pathologies.

Spatial biology studies have highlighted transcriptomic and proteomic differences in single cells, but they 1) cannot discern the temporal axis necessary to study translational dynamics or 2) are limited to very few cells at a time. Recent advances in single molecule detection assays, such as fluorescence in situ hybridization (FISH) and multiplex immunofluorescence, have enabled profiling of up to hundreds of molecules across thousands of cells ^9–13. We applied these methods on thousands of stimulated with a timed fixation method which stimulated batches of cells and fixed them after various times to increase throughput.


Materials and Methods:: Mouse fibroblast cells containing fluorescent NFκB-related reporters were cultured until confluent and plated on separate coverslips. All coverslips were stimulated with media containing tumor necrosis factor alpha (TNFα). After 0, 5, 10, and 20 mins, cells were fixed with 1.6% paraformaldehyde and labeled with subsequent cycles of RNAs and proteins related to NFκB signaling with single molecule FISH and multiplex immunofluorescence, respectively ^9,10. Cells were segmented with Cellpose ¹⁴ and partitioned into cytosol, nucleus, and nuclear membrane from the masks. To reduce the dimension of the dataset, we used tSNE ¹⁵ on the sum expression of each biomarker within each of the 3 subcellular compartments.


Results, Conclusions, and Discussions:: Results and Conclusions
We have successfully recapitulated stimulation dynamics among discrete groups of cells at 0, 5, 10, and 20 mins as evidenced by transition from cytosolic to nuclear p65 protein signal (Fig 1A). Subsequent fluorescent labeling with p65 RNA (Fig. 1B) reveals an inverse correlation between p65 protein and RNA expression levels (Fig. 1C), which may indicate a translational feedback mechanism. Furthermore, visual cell inspection illustrated 3 main areas of high p65 protein (3 levels of stimulation): the cytosol (unstimulated), nuclear membrane (quasi-stimulated), and nucleus (stimulated) (Fig. 1D). Classification of thousands of cells in each of these 3 categories confirmed more stimulated cells across time (Fig. 1E). Finally, dimensional reduction on p65 RNA and protein levels within each subcellular area for each cell was plotted with tSNE¹⁵ and colored by the stimulation level or time (Fig. 1E). The single cell embeddings indicate separation of some groups of cells with more clear partitions of stimulated cells and 20 min timepoint cells.

Discussion
Although a staple of the Central Dogma of biology, direct translation from RNA to protein has yet to be spatially observed. To more closely investigate this phenomenon, we cultured fibroblast cells with fluorescent NFκB reporters and used recent RNA detection techniques to spatially map individual p65 RNAs and proteins within each cell for thousands of cells. Our work highlights the single-cell variability and inverse correlation between RNA and protein regulation that may indicate a new translational regulation mechanism. Since dysregulation of NFκB is implicated in several disease states such as cancer, Alzheimer’s, arthritis, and others, our work may inform how translational regulation plays a role as well.

Acknowledgements (Optional): : Research reported in this study was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM142616.


References (Optional): : 1 Oeckinghaus, A. et al. Nat Immunol 12, 695–708 (2011)

2 Oeckinghaus, A. et al. Cold Spring Harb Perspect Biol 1, a000034 (2009)

3 Karin, M. Nature 441, 431–436 (2006)

4 Gerondakis, S. et al. Oncogene 18, 6888–6895 (1999)

5 Saw, P. E. et al. Sci China Life Sci 64, 22–50 (2021)

6 Koussounadis, A. et al. Sci Rep 5, 10775 (2015)

7 Truitt, M. L. et al. Nat Rev Cancer 16, 288–304 (2016)

8 Silvera, D. et al. Nat Rev Cancer 10, 254–266 (2010)

9 Choi, H. M. T. et al. Development 145, dev165753 (2018)

10 Venkatesan, M. et al. Sci Rep 13, 5374 (2023)

11 Xia, C. et al. Proceedings of the National Academy of Sciences 116, 19490–19499 (2019)

12 Giesen, C. et al. Nat Methods 11, 417–422 (2014)

13 Atmanli, A. et al. eLife 8, e49599 (2019)

14 Stringer, C. et al. Nat Methods 18, 100–106 (2021)

15 Kobak, D. et al. Nat Commun 10, 5416 (2019)