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TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 609 |
Dynamic
Regulation of Tec Kinase Localization in Membraneproximal
Vesicles of a T Cell Clone Revealed by Total Internal
Reflection Fluorescence and Confocal Microscopy |
Lawrence
P Kane, Simon C. Watkins |
tyrosine
kinases, lymphocyte activation, effector function, total internal
reflection fluorescence microscopy, early endosomal antigen 1 marker |
Glass bottom 35-mm cell culture
dishes (MatTek, Ashland, MA) were precoated overnight at 4 °C with antibodies to CD3 and
CD28 or LFA-1 and diluted to 10 g/ml in phosphate-buffered saline.
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total
internal reflection fluorescence and confocal microscopy |
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D10
cells |
| Abstract |
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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 Teccontaining
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- 1, and the early endosomal antigen 1 marker EEA1. |
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