Ruggero Pardi: Training Module 1

Direct Supervisor: Ruggero Pardi, MD
Location: Unit of Leukocyte Biology, Department of Functional Genomics
Descriptive title of the research activity: Structural Determinants of Cell Polarity in Migrating Leukocytes

Overall goals:

The overall goal of our research is to dissect the mechanisms underlying the asymmetric distribution of membrane components, including integrins, in directionally migrating leukocytes.  The central hypothesis is that integrin-containing molecular complexes, dynamically induced by motogenic stimuli, are required to establish and maintain polarity in vectorially migrating leukocytes.

Rationale and Significance:

Cell migration is a highly dynamic process featuring a defined sequence of tightly regulated steps. Directional migration involves the establishment and maintenance of a spatial and functional asymmetry of adhesion- and migration-related molecular components between the anterior (leading) and posterior (trailing) edges of the migrating cell (Lauffenburger and Horwitz, 1996; Ridley et al., 2003). How such coordinate redistribution of molecular components of the cell migration and signaling machinery is controlled and maintained over time is largely unknown.

Description of work:

To develop tools for quantitative imaging of integrin dynamics in migrating cells by time lapse video microscopy. To this aim, we will develop alternative tools based on the use of monomeric fluorescent proteins fused to integrins and to other relevant proteins in the process. We will initially create fusion constructs between nondimerizing, monomeric GFP, YFP, CFP and DsRED fluorescent proteins and both subunits of the aL/b2 integrin, and will assess their subcellular distribution and function in myeloid and non-myeloid cell lines such as CHO cells. To investigate the structural basis and physiological role of the dynamic inclusion of surface-expressed integrins in detergent-resistant membrane microdomains (DRM, or “rafts”). The aforementioned ectopic expression systems, as well as cell lines expressing the endogenous receptor, will be used to characterize, at the morphological and biochemical level, the structural determinants underlying the inclusion of integrins in DRMs.

Methodology:
Task 1: imaging active integrins in polarized cells through the generation of FP chimeric constructs (or BiFC reagents, see T. Kerppola et al.) based on proteins (e.g. talin head, calpain, GPI-AP, uPAR) known to associate with and promote the activation of integrins). Titration of the above protein levels to avoid overexpression-induced integrin activation. Detection: confocal MO, deconvolution MO and TIRM.
Task 2: imaging and quantitating integrin cytoskeletal associations in polarized cells through the creation of FP pairs of integrins and candidate cytoskeletal/signaling molecules. Engineer constructs to make them amenable to do FRET studies. Effect of Rho GTPases on integrin-cytoskeletal associations using RNAi to RhoA, Rac1/2.
Task 3: comparative proteomics analysis of integrin-associated proteins in polarized vs non polarized cells; role of the dynamic inclusion of integrins in lipid rafts (as judged by biochemical and morphological analysis) in establishing/maintaining polarization. Search for candidate molecules by co-ip and WB and for novel molecules by microsequencing.

--> Go to training module 2

or

--> Back to Ruggero Pardi's Home Page