Ph.D. Projects


 Chemokine-driven signaling leading to concurrent steps in leukocyte extravasation and interstitital migration.

    Leukocyte extravasation involves interdependent signaling pathways underlying the complex dynamics of firm adhesion, crawling and diapedesis. Signaling by agonist-bound chemokine receptors plays a central role in the above responses, but it is unclear how it contributes to the sustained and concurrent nature of such responses, given the rapid kinetics of chemokine-induced trimeric G protein coupling and homologous desensitization. We have recently identified a novel pathway triggered by agonist-induced CXCR2 receptors and regulating integrin function at multiple levels (Molteni, R. et al. Blood. 2009) The pathway to be investigated involves the G protein-coupled receptor (GPCR)-associated ubiquitous beta-arrestins and links chemokine stimulation, downstream of G protein coupling, to the sequential activation of leukocyte adhesion and migration via the modulation of the small GTPase Rap1 and the independent activation of the Phosphoinositide 3 kinase (PI3K) and ERK pathways. Most of the above pathways are selectively dependent on beta-arrestin 2, one of the two ubiquitous beta-arrestin isoforms, Goal of this project is the complete dissection of the above pathway and its validation in preclinical models of chronic inflammation. Several interdependent, measurable responses to motogenic stimulation will be investigated, particularly in cell types that are prominent effectors of chronic inflammatory responses. These issues will be accomplished by: a) developing novel reagents, screening approaches and biosensor probes to identify and characterize the signaling intermediates and effectors modulating beta-arrestin-dependent control of cell adhesion and migration by selected G protein-coupled receptors; b) using lentiviral vector-encoded shRNA sequences to genetically inactivate the identified signaling intermediates and effectors in diploid cell lines and bone marrow precursors, followed by reconstitution experiments in lymphomyeloid cells and primary cell lines, to validate their functional role both in vivo, by intravital microscopy, and in vitro using newly developed optical-based assays. The latter assays are being developed in collaboration with the Departments of Physics and Structural Engineering of the Polytechnical University in Milano.

Reference:

Molteni R, Crespo CL, Feigelson S, Moser C, Fabbri M, Grabovsky V, Krombach F, Laudanna C, Alon R, Pardi R. {beta}-arrestin 2 is required for the induction and strengthening of integrin-mediated leukocyte adhesion during CXCR2-driven extravasation. Blood. 2009 May 8. [Epub ahead of print]