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Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Simmons AJ, Banerjee A, McKinley ET, Scurrah CR, Herring CA, Gewin LS, Masuzaki R, Karp SJ, Franklin JL, Gerdes MJ, Irish JM, Coffey RJ, Lau KS - Mol. Syst. Biol. (2015)

Bottom Line: A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation.Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival.Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.

No MeSH data available.


Related in: MedlinePlus

DISSECT preserves native signal transduction during disaggregationComparison between early (p-ERK, p-C-JUN) and late (p-STAT3) signaling data generated from quantitative immunoblotting, conventional disaggregation method followed by flow cytometry, and DISSECT followed by flow cytometry. Immunoblotting data represent integrated intensity of an immunoblot band. Flow cytometry data represents the median intensity of single-cell distributions. Data scales are Z-score values derived from mean centering and variance scaling of each time course experiment (Appendix Fig S8). Error bars represent SEM from biological duplicates.
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fig02ev: DISSECT preserves native signal transduction during disaggregationComparison between early (p-ERK, p-C-JUN) and late (p-STAT3) signaling data generated from quantitative immunoblotting, conventional disaggregation method followed by flow cytometry, and DISSECT followed by flow cytometry. Immunoblotting data represent integrated intensity of an immunoblot band. Flow cytometry data represents the median intensity of single-cell distributions. Data scales are Z-score values derived from mean centering and variance scaling of each time course experiment (Appendix Fig S8). Error bars represent SEM from biological duplicates.

Mentions: A major challenge for exploring signaling heterogeneity in epithelial tissues with cytometry-based methods is the requirement of single-cell suspensions. Previous attempts to probe epithelial signaling involved stimulation experiments on single epithelial cells that were already dissociated and outside of their native contexts (Lin et al, 2010). To study single-cell signaling in the in situ epithelial context, we first tested whether a single-cell disaggregation procedure used routinely for flow sorting epithelial cells (Magness et al, 2013) (which we referred to as “the conventional method”) preserves native signaling in single-cell suspensions. Briefly, the intestinal epithelium was mechanically retrieved after the intestine was acquired, washed, and longitudinally opened. The epithelium was then digested enzymatically (∼10 min) and then filtered into a single-cell suspension. A standard fix-perm procedure for phospho-flow was then performed, followed by phospho-specific antibody staining and cytometry analysis (Krutzik et al, 2004). Quantitative immunoblotting analysis on fresh intestinal tissue lysates was used as a positive control. A head-to-head assessment using the same antibodies was performed by comparing median intensities from single-cell flow cytometric data to integrated intensities of bands from immunoblots, which reflect cell averages in tissue lysates. This comparison demonstrated that signal transduction induced by TNF-α was not maintained with the conventional disaggregation method, as assessed by both early (p-ERK1/2, p-C-JUN) and late (p-STAT3) signals (FigEV2). A previous study suggested that signaling perturbations from tryptic disaggregation can be eliminated by performing digestion in live cells at low temperatures (Abrahamsen & Lorens, 2013). We tested the effect of enzymatic digestion by performing low-yield single-cell disaggregation on live tissues (Appendix Fig S1A), using gentle mechanical dissociation without any enzymes; however, signal transduction was still not preserved (Appendix Fig S1B). Disaggregation of an intact epithelium into single cells perturbs components of epithelial cell junctions that play many roles in signaling modulation. Such disruption in live tissue may dynamically alter signaling pathways and produce experimental artifacts.


Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Simmons AJ, Banerjee A, McKinley ET, Scurrah CR, Herring CA, Gewin LS, Masuzaki R, Karp SJ, Franklin JL, Gerdes MJ, Irish JM, Coffey RJ, Lau KS - Mol. Syst. Biol. (2015)

DISSECT preserves native signal transduction during disaggregationComparison between early (p-ERK, p-C-JUN) and late (p-STAT3) signaling data generated from quantitative immunoblotting, conventional disaggregation method followed by flow cytometry, and DISSECT followed by flow cytometry. Immunoblotting data represent integrated intensity of an immunoblot band. Flow cytometry data represents the median intensity of single-cell distributions. Data scales are Z-score values derived from mean centering and variance scaling of each time course experiment (Appendix Fig S8). Error bars represent SEM from biological duplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631206&req=5

fig02ev: DISSECT preserves native signal transduction during disaggregationComparison between early (p-ERK, p-C-JUN) and late (p-STAT3) signaling data generated from quantitative immunoblotting, conventional disaggregation method followed by flow cytometry, and DISSECT followed by flow cytometry. Immunoblotting data represent integrated intensity of an immunoblot band. Flow cytometry data represents the median intensity of single-cell distributions. Data scales are Z-score values derived from mean centering and variance scaling of each time course experiment (Appendix Fig S8). Error bars represent SEM from biological duplicates.
Mentions: A major challenge for exploring signaling heterogeneity in epithelial tissues with cytometry-based methods is the requirement of single-cell suspensions. Previous attempts to probe epithelial signaling involved stimulation experiments on single epithelial cells that were already dissociated and outside of their native contexts (Lin et al, 2010). To study single-cell signaling in the in situ epithelial context, we first tested whether a single-cell disaggregation procedure used routinely for flow sorting epithelial cells (Magness et al, 2013) (which we referred to as “the conventional method”) preserves native signaling in single-cell suspensions. Briefly, the intestinal epithelium was mechanically retrieved after the intestine was acquired, washed, and longitudinally opened. The epithelium was then digested enzymatically (∼10 min) and then filtered into a single-cell suspension. A standard fix-perm procedure for phospho-flow was then performed, followed by phospho-specific antibody staining and cytometry analysis (Krutzik et al, 2004). Quantitative immunoblotting analysis on fresh intestinal tissue lysates was used as a positive control. A head-to-head assessment using the same antibodies was performed by comparing median intensities from single-cell flow cytometric data to integrated intensities of bands from immunoblots, which reflect cell averages in tissue lysates. This comparison demonstrated that signal transduction induced by TNF-α was not maintained with the conventional disaggregation method, as assessed by both early (p-ERK1/2, p-C-JUN) and late (p-STAT3) signals (FigEV2). A previous study suggested that signaling perturbations from tryptic disaggregation can be eliminated by performing digestion in live cells at low temperatures (Abrahamsen & Lorens, 2013). We tested the effect of enzymatic digestion by performing low-yield single-cell disaggregation on live tissues (Appendix Fig S1A), using gentle mechanical dissociation without any enzymes; however, signal transduction was still not preserved (Appendix Fig S1B). Disaggregation of an intact epithelium into single cells perturbs components of epithelial cell junctions that play many roles in signaling modulation. Such disruption in live tissue may dynamically alter signaling pathways and produce experimental artifacts.

Bottom Line: A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation.Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival.Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.

No MeSH data available.


Related in: MedlinePlus