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Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow
Published on: August 14, 2014
Endothelial cell cortactin phosphorylation by Src contributes to polymorphonuclear leukocyte transmigration in vitro
Lin Yang1, Jennifer R Kowalski, Xi Zhan
1Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Circulation Research
|December 31, 2005
Summary
Src-induced cortactin phosphorylation in endothelial cells is crucial for polymorphonuclear leukocyte (PMN) transmigration. This process regulates PMN movement across the endothelium, not adhesion.
Area of Science:
- Immunology
- Cell Biology
- Vascular Biology
Background:
- Leukocyte transendothelial migration is vital for immune response but its mechanisms are unclear.
- Cortactin, a cytoskeletal regulator, is phosphorylated by Src tyrosine kinases.
- Previous work linked Src-mediated cortactin phosphorylation to adhesion molecule clustering.
Purpose of the Study:
- To investigate the role of Src-induced cortactin phosphorylation in endothelial cells during polymorphonuclear leukocyte (PMN) transmigration.
- To elucidate the specific contribution of cortactin phosphorylation to PMN movement across the endothelium.
Main Methods:
- Utilized an in vitro flow model with human umbilical vein endothelium (HUVEC) monolayers.
- Employed Src kinase inhibitors (PP2, SU6656) and cortactin knockdown via small interfering RNA (siRNA).
- Performed live cell imaging of GFP-tagged cortactin and analyzed PMN transmigration and adhesion.
Main Results:
- Src inhibition significantly reduced PMN transmigration (45-36%).
- Cortactin knockdown impaired transmigration, a phenotype rescued by wild-type cortactin reexpression.
- Tyrosine phosphorylation of cortactin was essential; a non-phosphorylatable mutant (cortactin3F) failed to rescue transmigration and partially blocked it.
Conclusions:
- Src-mediated tyrosine phosphorylation of cortactin is a key regulator of PMN transmigration across the endothelium.
- This phosphorylation event specifically influences PMN migration at endothelial cell junctions, not initial adhesion.
- Cortactin phosphorylation by Src family kinases provides a novel target for modulating inflammatory cell trafficking.
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