A multidisciplinary approach to the study of T cell migration.
Active T cell locomotion depends on efficient repeated cycles of integrin receptor/ligand interactions mediating cell adhesion and detachment, intracellular signaling cascades orchestrating posttranslation modifications of interacting proteins, dynamic reassembly of participating cytoskeletal elements, and structural support of associated scaffolding molecules. Using an integrated approach based on novel cutting edge technologies of live cell imaging, cell transfection, proteomics, and nanotechnology, we provide here a detailed characterisation of crucial mechanisms involved in LFA-1 integrin-mediated T cell migration. Polarization and phenotypic changes associated with LFA-1-triggered T cell locomotion is largely dependent on the intact functioning of the microtubule cytoskeleton. Experiments utilizing 4-D (3-D over time) confocal live imaging of T cells, microinjected with fully functional constructs encoding protein kinase C beta (PKC-beta) isoenzyme tagged with enhanced green fluorescent protein (GFP), elucidate that LFA-1-induced activation is associated with translocation of PKC-beta to sites associated with centrosomes and tubulin cytoskeleton in locomotory T lymphocytes. We also provide here a characterization of a novel microfluidics-based multichannel platform enabling detailed analysis of leukocyte adhesion and migration under regulated shear stress conditions. Using precision machined surfaces, we demonstrate that the substrate topography can influence the motile response of the two different T cell types in different ways, and this can be quantified in terms of specified motility parameters. Finally, using an original in situ immunoprecipitation method, in which LFA-1 antibodies are utilized to induce intracellular association of proteins in the cytoskeletal/signaling complex, we demonstrate that this complex includes a number of structural and signaling proteins, which have been identified by 2-D electrophoresis and MALDI-TOF protein sequencing.
Long
Aideen
A
Department of Biochemistry, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland.
Mitchell
Siobhan
S
Kashanin
Dmitry
D
Williams
Vivienne
V
Prina Mello
Adriele
A
Shvets
Igor
I
Kelleher
Dermot
D
Volkov
Yuri
Y
D002448:Cell Adhesion; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D016023:Integrins / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology; D008214:Lymphocytes / Q000276:immunology; D008954:Models, Biological; D036103:Nanotechnology; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D016023:Integrins; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C