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

Lars Zimmermann, Wolfgang Paster, Julian Weghuber, Paul Eckerstorfer, Hannes Stockinger, Gerhard J. Schütz

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

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 membraneproximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ∼400 ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated at 50 s; together with the mobility of 0. μm2/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation-deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5.

Original languageEnglish
Pages (from-to)6063-6070
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number9
DOIs
Publication statusPublished - 26 Feb 2010

Keywords

  • Cell Membrane
  • Cysteine/metabolism
  • Cytosol
  • Diffusion
  • Humans
  • Jurkat Cells
  • Kinetics
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism
  • Microscopy, Fluorescence
  • Palmitates/metabolism
  • Protein Processing, Post-Translational
  • Protein Transport
  • Signal Transduction
  • T-Lymphocytes/metabolism

Fingerprint Dive into the research topics of 'Direct observation and quantitative analysis of Lck exchange between plasma membrane and cytosol in living T cells'. Together they form a unique fingerprint.

Cite this