Direct observation and quantitative analysis of Lck exchange between plasma membrane and cytosol in living T cells.

ABSTRACT: Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently labeled Lck, the key Src family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membrane-pro

SUBMITTER: Zimmermann L 

PROVIDER: S-EPMC2825400 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature