title: A quantitative assessment of costimulation and phosphatase activity on microclusters in early T cell signaling.
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.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: J Joris
authors.initials: JJ
authors.lastname: Witsenburg
authors.forename: Heike
authors.initials: H
authors.lastname: Glauner
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Müller
authors.forename: Johannes M M
authors.initials: JM
authors.lastname: Groenewoud
authors.forename: Günter
authors.initials: G
authors.lastname: Roth
authors.forename: Frank-Dietmar
authors.initials: FD
authors.lastname: Böhmer
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D018106:CD28 Antigens / Q000378:metabolism; D061025:Costimulatory and Inhibitory T-Cell Receptors / Q000378:metabolism / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006801:Humans; D019169:Jurkat Cells; D051966:Phospholipase C gamma / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D018106:CD28 Antigens; D061025:Costimulatory and Inhibitory T-Cell Receptors; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D010744:Phosphoric Monoester Hydrolases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D051966:Phospholipase C gamma