Department of Physiology, McGill University, Montreal, Quebec, Canada
 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ONGOING PROJECTS



Photo taken by Pierre Guzzo for the Association québécoise de la fibrose kystique.


The work in this lab is focused on epithelial chloride secretion and cell signaling, and how these may be altered in disease states such as cystic fibrosis and interstitial cystitis. The projects address important questions using molecular biology, electrophysiology, or imaging approaches (or some combination of these.

 1) CFTR structure/function: We want to understand the functional significance of nucleotide binding and hydrolysis at each nucleotide binding domain. Another major interest is understanding how CFTR is regulated by phosphorylation of its R domain. For these studies we use mutagenesis of nucleotide binding domains, consensus phosphorylation sites, phosphopeptide mapping, and studies of fusion proteins that contain the regulatory and nucleotide binding domains. The role of PKC is also being addressed by Ussing chamber studies and trafficking assays. The phosphatase that regulates the CFTR channel is of interest since it has not yet been identified at the molecular level and is a potential therapeutic target for the treatment of cystic fibrosis. Co-immunoprecipitations of co-expressed PP2C isoforms/splice variants and proteomics are being used to identify CFTR-associated phosphatase activity.

2) Interactions with other proteins: CFTR interacts with many proteins during its processing, trafficking and function at the cell surface. To identify those partners and examine their interactions biochemically and physiologically, a biotinylatable CFTR mutant has been generated with an extracellular recognition sequence for bacterial biotin ligase that enables specific biotinylation of CFTR at the cell surface. Biotinylated CFTR and its associated proteins are being copurified on streptavidin beads in vitro for proteomics, and labelled with fluorescent streptavidin for confocal microscopy studies on living cells. Image correlation spectroscopy of fluorescently tagged molecules is being used to measure CFTR mobility and study the effects of mutations and other maneuvers that may disrupt protein-protein interactions. Other techniques that exploit various tagged CFTR constructs are under development.

3) Autocrine and paracrine signaling by ATP and other epithelia-derived factors: ATP is released by most cells and serves as an important extracellular signaling molecule. We are interested in the mechanism of ATP release and how it is regulated by mechanical and other stimuli. ATP release regulates fluid and mucus secretion in the airways, and excessive ATP release by epithelial cells of the urinary bladder has been implicated in a painful disorder called interstitial cystitis. We use wild-type and mutated firefly luciferases, luminometry, and new cell culture and imaging methods to study ATP release by epithelial cells from airway submucosal glands and urinary bladder under conditions that simulate control and disease states. Our studies of epithelial secretion are being extended to freshly isolated tissues.

4) Native tissue studies: New projects that investigate airway submucosal gland from normal and CF tissues using fluid transport assays, amperometry, and imaging of fluorescent constructs are presently underway.

This page was last edited on 09 February, 2011