Cell membranes contain localized regions of specialized lipid and protein composition known as membrane microdomains. Recently, the properties of a class of membrane microdomains termed lipid rafts have become of great interest due to their proposed role in membrane trafficking, cell signaling, and the entry and exit of pathogens from cells. Commonly defined as cholesterol and sphingolipid-enriched domains, lipid rafts are thought to regulate protein function by concentrating some proteins into rafts, while segregating others in non-raft regions of the membrane. However, many of the fundamental properties of lipid rafts remain unknown, including the mechanism(s) by which specific proteins are targeted to lipid rafts. A commonly cited model for the association of proteins with lipid rafts is drawn by analogy to the partitioning of lipid probes in mixtures of liquid-ordered and liquid-disordered lipid phases. We hypothesize that this partitioning model is insufficient to explain the behavior of raft proteins in cells. Instead, we postulate that raft proteins are targeted to actively maintained domains in a cholesterol-dependent manner, and that the mode of membrane anchorage is a major determinant of the mechanism of raft association for any given protein. To test this hypothesis, we propose to investigate the mechanisms that govern the raft association of three commonly studied raft proteins: cholera toxin B-subunit (a glycolipid-binding protein), hemagglutinin (a transmembrane protein), and GFP-GPI (a glycosylphosphatidylinositol-anchored protein). To do so, we will perform biophysical measurements of raft- and non-raft proteins in living cells in combination with computer simulations of raft formation.
The specific aims of these studies are (1) to determine if the sub-micron distribution and diffusional mobility of three representative raft proteins are consistent with an actively maintained model for raft formation;(2) to determine if different types of raft proteins co-localize or compete for residence in the same rafts;and (3) to generate in silico simulations of lipid raft assembly mechanisms. Completion of these studies will provide new information regarding the mechanisms that underlie targeting of proteins to rafts in cells. Such information is critical to the eventual design of therapies targeted at interfering with or enhancing lipid raft function to improve human health. These studies will also contribute to our long- term goal of understanding how membrane structure regulates cellular functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073846-05
Application #
7826968
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2006-05-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
5
Fiscal Year
2010
Total Cost
$258,227
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Day, Charles A; Kenworthy, Anne K (2015) Functions of cholera toxin B-subunit as a raft cross-linker. Essays Biochem 57:135-45
Day, Charles A; Baetz, Nicholas W; Copeland, Courtney A et al. (2015) Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis. Traffic 16:572-90
Hanson, Caroline A; Drake, Kimberly R; Baird, Michelle A et al. (2013) Overexpression of caveolin-1 is sufficient to phenocopy the behavior of a disease-associated mutant. Traffic 14:663-77
Kraft, Lewis J; Kenworthy, Anne K (2012) Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4B(C74A) in live cells using Fo?rster resonance energy transfer and fluorescence recovery after photobleaching. J Biomed Opt 17:011008
Day, Charles A; Kenworthy, Anne K (2012) Mechanisms underlying the confined diffusion of cholera toxin B-subunit in intact cell membranes. PLoS One 7:e34923
Chinnapen, Daniel J-F; Hsieh, Wan-Ting; te Welscher, Yvonne M et al. (2012) Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1. Dev Cell 23:573-86
Kang, Minchul; Day, Charles A; Kenworthy, Anne K et al. (2012) Simplified equation to extract diffusion coefficients from confocal FRAP data. Traffic 13:1589-600
Day, Charles A; Kraft, Lewis J; Kang, Minchul et al. (2012) Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP). Curr Protoc Cytom Chapter 2:Unit2.19
Lapierre, Lynne A; Ducharme, Nicole A; Drake, Kimberly R et al. (2012) Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells. Exp Cell Res 318:103-13
Kang, Minchul; DiBenedetto, Emmanuele; Kenworthy, Anne K (2011) Proposed correction to Feder's anomalous diffusion FRAP equations. Biophys J 100:791-2; author reply 793-4

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