FELLOWSHIPS
We gratefully acknowledge support of post-doctoral fellows from the following organizations:
GRANTS
U.S.P.H.S. Grant GM-22942
"Intracellular Translocation and Metabolism of Lipids."
Total project period: 3/1/79 through 2/28/11
Glycosphingolipids (GSLs) play important roles in a wide variety of cell functions, including cell-cell interactions, cell growth and differentiation, and signal transduction. GSLs can interact with cholesterol to form membrane microdomains, and data from many studies suggest that the plasma membrane (PM) GSL and cholesterol composition may be tightly regulated. To achieve this regulation, cells must balance complex processes underlying the intracellular transport of GSLs with their synthesis and degradation. In the present application, four major projects will be pursued to better understand this regulation. We will:
- Study the molecular determinants of GSLs that result in their selective endocytosis via a clathrin-independent, caveolar-like process in many cell types. Particular emphasis will be placed on the importance of lipid-lipid vs. lipid-glycoprotein interactions at the PM in determining the internalization mechanism. We will also study the involvement of caveolin-1 in this endocytic process and the nature of GSL internalization in cells lacking caveolae;
- Study the intracellular sorting of internalized SLs for recycling. Of particular interest is whether lipids internalized via coated pits recycle to the PM with similar rates as lipids internalized via caveolae, and whether they use the same protein machinery (e.g., rabs4 and 11). We will also investigate the distribution of GSL analogs into microdomains within early endosomes, and the rapid intermixing (and further sorting) of GSLs internalized via caveolae vs markers for the clathin pathway;
- Test the hypothesis that PM GSL endocytosis and recycling are regulated by GSL synthesis and delivery to the PM, by modulating GSL synthesis or PM lipid composition;
- Continue our molecular and functional studies of glucosylceramide synthase which catalyzes the first glycosylation step in the formation of most higher-order GSLs, and is an important regulator of GSL homeostasis.
The proposed studies are particularly timely because of many recent developments in the field pertaining to the organization of GSLs in membranes and to their emerging functional roles. A greater understanding of the caveolar-like endocytic pathway is also extremely important because the same or similar pathways are utilized for cell entry and intracellular delivery of some bacterial toxins, viruses, and bacteria.
U.S.P.H.S. Grant GM-60934
"Cholesterol and Sphingolipid Perturbation of Membrane Traffic in Human Disease"
Total project period: 12/22/00 through 11/30/12
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cAMP-dependent chloride channel CF transmembrane conductance regulator (CFTR). Some missense mutations, including deletion of phenylalanine 508 (ΔF508), the most common mutation in CF disease, cause retention of the protein in the ER and premature degradation. As a result there is a significant reduction in functional CFTR in the plasma membrane of airway epithelial cells leading to defective chloride secretion, hyper-absorption of sodium, and other changes that reduce the capacity of cilia to clear bacteria from the airways. Recently several groups, including our own, have shown that cholesterol and sphingolipids (SLs) accumulate in CF cells, similar to that seen in various sphingolipid storage diseases. These findings and key preliminary data led to our central hypothesis that increased levels of cholesterol and SLs exacerbate the symptoms of CF by interfering with normal membrane transport processes and CFTR function. In this application, three major projects will be pursued to test this hypothesis:
- We will focus on over-expression of Rab1 and Rab2, two GTPases involved in ER to Golgi transport, as a tool for enhancing the delivery of ΔF508 to the plasma membrane. These studies will provide a new method for increasing ΔF508-CFTR at the plasma membrane and allow us to carry out studies on the underlying mechanism for this enhanced delivery as well as the function of the mutant protein at the cell surface.
- We will study the effects of SLs and cholesterol on endocytosis, recycling, and function of WT- and ΔF508-CFTR since these lipids can significantly affect membrane transport as well as the microenvironment of other membrane proteins. Based on preliminary data showing reduced cilia in ΔF508-CFTR cells, we will also study the effect of alterations in cholesterol on (i) the distribution of cilia in cells expressing wild type or mutant CFTR, (ii) modulation of RabGTPases involved in ciliogenesis, and (iii) the distribution of cholesterol binding proteins that are normally associated with cilia.
- We will study the mechanism underlying elevation of SLs and cholesterol in mutant CFTR cells to learn whether the absence of CFTR function or the presence of a misprocessed mutant CFTR in the distal secretory pathway is responsible for lipid storage. We will also investigate the causal relationships between mutant CFTR expression, elevated lysosomal pH, and lipid storage. We will also use methods developed in the previous grant period and in preliminary studies (e.g., Caveolin-1 knock-down) to deplete elevated cholesterol and SLs from cells, in an attempt to reverse various aspects of the ΔF508 phenotype.
Together these proposed experiments should increase our understanding of the connection between mutant CFTR and lipid storage and may also provide additional approaches for treatment of CF.
