Lck is Randomly Distributed on the T Cell Plasma Membrane

Abstract

The spatial organization of signaling components on the T cell plasma membrane is generally believed to regulate T cell function. During activation, local immobilization in so-called microclusters and subsequent rearrangement of key components of the T cell signaling machinery results in down-stream signaling and T cell activation1. Using super-resolution microscopy, a number of reports have proposed the existence of nanoscopic clusters of T cell signaling components2-4, suggesting that local enrichment of signaling molecules is an underlying regulatory mechanism of T cell signaling. Here, we investigate more in detail the nanoscopic organization of Lck in Jurkat T cells. Contrary to a previous report that described Lck nano-clusters using PALM, we find both in PALM and dStorm experiments that Lck is homogeneously distributed on the T cell plasma membrane. We develop a new approach to analyze apparent nano-clustering in PALM and dStorm data of fixed cell samples. By titrating the labeling density, we calculate cluster sizes and localization densities in clusters to distinguish random from clustered spatial distributions of molecules. Moreover, we also use two-color labeling in combination with nearest neighbor distance analysis to characterize the spatial distribution of molecules. Our data suggest that Lck activity and hence T cell activation cannot depend on the formation of nanoscopic clusters. Rather, our data support the view that Lck is randomly distributed on the T cell plasma membrane and is specifically recruited to signaling hotspots during T cell activation. Funding: Austrian Science Fund/FWF P 27941-B28 1. Dustin M.L. (2014) Cancer Immunol Res 2. Owen D.M. et al (2012) Nat Commun 3. Rossy J. et al (2013) Nat Immunol 4. Lillemeier B.F.F. et al (2010) Nat Immunol