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Signalling protein complexes isolated from primary human skin-resident T cells can be analysed by Multiplex IP-FCM.

ABSTRACT: Studying signal transduction in skin-resident T cells (sr-T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr-T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP-FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr-T cells isolated from small clinical samples provide sufficient material for mIP-FCM-based analysis of signalling-induced protein complexes. We propose that this strategy may be useful for gaining improved mechanistic insight of sr-T cell signal transduction associated with dermatological disease.

SUBMITTER: Smith SE

PROVIDER: S-EPMC4003868 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Signalling protein complexes isolated from primary human skin-resident T cells can be analysed by Multiplex IP-FCM.

Smith Stephen E P SE Neier Steven C SC Davis Tessa R TR Pittelkow Mark R MR Gil Diana D Schrum Adam G AG

Experimental dermatology 20140401 4

Studying signal transduction in skin-resident T cells (sr-T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr-T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP-FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr-T ...[more]

PMID: 24588717

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Project description:BackgroundTo understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success.Methodology/principal findingsWe use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally.Conclusions/significanceThe quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network.

Dataset Information

Signalling protein complexes isolated from primary human skin-resident T cells can be analysed by Multiplex IP-FCM.

Publications

Signalling protein complexes isolated from primary human skin-resident T cells can be analysed by Multiplex IP-FCM.

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