Ligation of CD3 triggers transmembrane proximity between LFA-1 and cortical microfilaments in a cytotoxic T cell clone derived from tumor infiltrating lymphocytes: a quantitative resonance energy transfer microscopy study.
We have explored the transmembrane associations of leukocyte function associated antigen-1 (LFA-1) in response to T cell receptor ligation using resonance energy transfer (r.e.t.) microscopy to detect receptor to microfilament proximity. R.e.t. was detected using both imaging and photon counting techniques. T cells were labeled with fluorescein-conjugated F(ab')2 fragments of an anti-LFA-1 monoclonal antibody. Cells were incubated at 37 degrees C on unmodified glass surfaces and surfaces coated with anti-CD3 or anti-H-9 antibodies. Microfilaments of fixed cells were labeled with rhodamine-phalloidin. R.e.t. was not affected on unmodified (blank) or irrelevant antibody-treated (H-9) surfaces. However, both fluorescence images and photon count rates were significantly enhanced when cells bound to anti-CD3-coated surfaces. This enhancement was not due to a general effect of T cell activation on transmembrane cytoskeletal proximity since CD45-phalloidin r.e.t. was not affected by CD3 ligation. These experiments provide direct physical evidence that ligation of the CD3 complex specifically increases the proximity of LFA-1 and microfilaments, which may be relevant to T cell mediated adherence reactions.
Poo
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Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
Fox
B A
BA
Petty
H R
HR
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D017252:CD3 Complex / Q000032:analysis / Q000276:immunology / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002999:Clone Cells; D003599:Cytoskeleton / Q000648:ultrastructure; D004735:Energy Transfer; D008026:Ligation / Q000379:methods; D016169:Lymphocyte Function-Associated Antigen-1 / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D016246:Lymphocytes, Tumor-Infiltrating / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D051379:Mice; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence; D010590:Phalloidine; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D016169:Lymphocyte Function-Associated Antigen-1; D010590:Phalloidine