Dynamic regulation of Tec kinase localization in membrane-proximal vesicles of a T cell clone revealed by total internal reflection fluorescence and confocal microscopy.
Tec family tyrosine kinases are key regulators of lymphocyte activation and effector function. Several Tec family kinases (Tec, Itk, Rlk/Txk) are expressed in T cells, but it is still not clear to what degree these are redundant or have unique functions. We recently demonstrated that Tec alone, among the Tec kinase family members examined, can induce nuclear factor of activated T cell-dependent transcription. This unique functional characteristic correlated with a unique pattern of subcellular localization, as Tec (but not other family members) was found in small vesicles, the appearance of which requires signaling through the T cell receptor for antigen. Here we report on our studies of these Tec-containing structures in live T cells, using total internal reflection fluorescence microscopy. With this technique, we showed that, in live T cells, the Tec vesicles are located at the plasma membrane, the vesicles are unique to Tec (and not the related kinase Itk), and their formation and maintenance require T cell receptor signaling through Src family kinases and PI 3-kinase. Finally, we have imaged isolated T cell membranes by confocal microscopy, confirming the membrane-proximal location of Tec vesicles, as well as demonstrating overlap of these vesicles with the tyrosine kinase Lck, the Tec substrate PLC-gamma1, and the early endosomal antigen 1 marker EEA1.
Kane
Lawrence P
LP
Department of Immunology, BST E-1056, University of Pittsburgh, Pittsburgh, PA 15261, USA. lkane@pitt.edu
Watkins
Simon C
SC
D000818:Animals; D001324:Autoantigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma; D010957:Plasmids / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D033921:Vesicular Transport Proteins; D019061:src-Family Kinases / Q000378:metabolism
D001324:Autoantigens; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma