Abstract
Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.
Highlights
Cell adhesion to the extracellular matrix (ECM)1 is mediated predominantly by the integrin family of heterodimeric transmembrane glycoproteins, which are concentrated at discrete sites
In a search for molecules that interact with paxillin LIM domains, a yeast two-hybrid screen was performed with the C-terminal half of paxillin fused to LexA as a bait
Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter molecule interacting with several structural and signaling proteins associated with focal adhesions and the actin cytoskeleton [3]
Summary
Reagents and Antibodies—Fibronectin (FN) and poly-L-lysine were obtained from Sigma. ICAM-1-Fc was obtained as described [17]. A lysate of T lymphoblasts (150 ϫ 106 cells) was prepared in ice-cold lysis buffer A (20 mM Hepes, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride). Either 2 ϫ 104 T lymphoblasts or monocytes were incubated at 37 °C for 1 h, on coverslips coated with 5 g/ml ICAM-1Fc protein or 10 g/ml FN and blocked with 1% bovine serum albumin, or coated with 0.01% poly-L-lysine or a suspension of peripheral blood lymphocytes was cytocentrifuged onto glass slides. Cells were stained with specific mAb or polyclonal antibodies, and after washing, were incubated with Cy2-labeled rabbit anti-mouse IgG and/or Cy3-labeled goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). Images of cellular sections were acquired every 0.5 m
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