NDLI: A Quantitative Assessment of Costimulation and Phosphatase Activity on Microclusters in Early T Cell Signaling

Content Provider	PubMed Central
Author	Joris, Witsenburg J. Glauner, Heike Müller, Jörg P. Groenewoud, Johannes M. M. Roth, Günter Böhmer, Frank-dietmar Adjobo-hermans, Merel J. W. Brock, Roland
Editor	Bansal, Geetha P.
Copyright Year	2013
Abstract	T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.
Related Links	http://dx.doi.org/10.1371/journal.pone.0079277
Starting Page	79277
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	10
Volume Number	8
Language	English
Publisher	Public Library of Science
Publisher Date	2013-10-30
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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