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Direct Supervisor: Francisco Sanchez-Madrid, MD
Location: Servicio de Inmunología. Departamento de Medicina. Hospital Universitario de la Princesa.
Descriptive title of research activity: Regulatory mechanisms and functional role of polarized adhesion and chemokine receptors orchestrating leukocyte migration
Overall goals: We aim at dissecting the functional role of dynamic interactions among vascular endothelial adhesion molecules (ICAMs/VCAM-1/JAMs, integrin/tetraspanin microdomains), regulatory molecules (PKCII and ), and cytoskeletal components (ERM proteins) that orchestrate leukocyte adhesion and transendothelial migration.
Rationale and significance: Endothelial receptors VCAM-1 and ICAM-1 participate together with actin cytoskeleton in the formation of an endothelial actin-rich docking structure that anchors the adherent leukocytes during transendothelial migration. Tetraspanins form protein-based microdomains in the plasma membrane that include several transmembrane proteins such as integrins or the Immunoglobulin superfamily proteins involved in intercellular adhesion and migration. Tetraspanins are components of the docking structure, through lateral associations with both ICAM-1 and VCAM-1, that have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration. The role of tetraspanins in E-selectin- and VCAM-1-mediated rolling, and their posible involvement in leukocyte transmigration will be investigated under flow conditions. Other interactions of CXCR4 with cytoskeleton have been identified and will be explored.
Description of work and methodology: The work our group is addressing involves the understanding of the cellular and molecular mechanisms regulating the interactions of chemokine and adhesion receptors with cytoskeletal components and signaling pathways that coordinate the proper adhesion and migration. Most of the experimental approaches undertaken in the laboratory involve molecular dynamic studies with fluorescent-tagged molecules and confocal time-lapse videomicroscopy in living cells.
1: Endothelial docking structure for adherent and transmigrating leukocytes: functional, structural and signaling components and regulation by tetraspanin microdomains: A) Analysis of VCAM-1 and ICAM-1 membrane dynamics at the docking structure using fluorescence recovery after photobleaching (FRAP) technique; B) Study of VCAM-1 and ICAM-1 co-presentation on the apical cell surface of endothelium independently of ligand binding or cytoskeletal anchorage; C) Role of tetraspanin microdomains in regulating rolling (selectin- and VCAM-1 dependent) and diapedesis (JAMs-, and cadherin-dependent) steps; D) Involvement of classic and novel PKCs (specifically PKCII and PKC in the disassembly of the endothelial docking structure to allow the progression of leukocyte extravasation during inflammation. Role of tetraspanin microdomains as anchoring platforms for the recruitment of PKCs to the endothelial docking structure; E) Functional association of metalloprotease MT1-MMP with tetraspanins.
2: Regulatory role of the interactions of chemokine receptors and cytoskeleton during chemokine-directed lymphocyte migration: A) Functional role of Myosin Heavy Chain II interaction with Chemokine receptor CXCR-4 in receptor endocytosis and molecular complexes involved; B) Characterization of cytoskeletal interactions of debrin with chemokine receptor CXCR-4; C) Functional role of adapter molecule GIT1 in chemotaxis.
Methodology: To perform dynamic studies of leukocyte transendothelial migration under flow conditions, we will use a parallel flow chamber coupled to a multidimensional fluorescence microscope unit that allows the rapid capture of high resolution images. VCAM-1 and ICAM-1 will be tracked during the formation of the docking structure around adherent leukocytes by microinjection of primary endothelial cells (HUVEC) with human ICAM-1 and VCAM-1 cDNAs coupled to GFP. Time-lapse images will be processed with a deconvolution software to obtain three-dimensional information of the docking structures formed. To analyse whether VCAM-1 and ICAM-1 exhibit a dynamic molecule interchange at the docking structure, we will use a laser scanning spectral confocal microscope to perform FRAP. The functional relevance of tetraspanin microdomains on endothelial intercellular adhesion molecules (VE-cadherin/catenins, and JAMs) will be assessed by using either siRNAs for endothelial tetraspanins or recombinant inhibitory peptides containing the LEL of tetraspanins. We will also use the mAbs, cDNAs, cell lines derived from KO mice of VE-Cadherin or JAM and related constructs.
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