This project aims to understand biomembrane structure and function with the aid of a recent lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. This permits preparation of lipid vesicles mimicking natural membranes closely in terms of both lipid composition and, for the first time, lipid asymmetry, the difference in the lipid composition in the inner and outer lipid layers characteristic of many natural membranes. The method is also being extended to control of lipid composition in living cells. These methods are being applied to solve long-standing issues of membrane domain formation. We and collaborator Dr. Deborah Brown proposed in 1994 what remains the working model in the field: cell membrane domains form due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. Although such domains are readily observed in artificial lipid vesicles, and careful studies document their presence in living cells, domains remain controversial and poorly characterized. This project will use lipid exchange to overcome roadblocks to progress in studies of membrane domains. First, studies using asymmetric lipid vesicles that mimic cell membranes much more closely than the symmetric vesicles employed in the past will define the rules governing domain properties and formation by lipids and proteins. This includes testing the long-standing hypothesis that lipid-induced signal transduction across membranes can result from coupling between physical properties of lipids in the inner and outer lipid layers of a membrane. Knowledge gained from these studies will reveal how to manipulate lipids and proteins in cells to control domain formation and protein association with domains, and thus how to explore their function. Second, domains will be studied with more tractable living systems and methods. We found that the cholesterol-containing bacterium Borrelia burgdorferi, the causative agent of Lyme disease, has domain size sufficient for visualization by electron microscopy and facile fluorescence domain detection using FRET, plus accessibility to altering sterol chemical structure to allow controlled modulation of domain formation. This made it possible to unequivocally identify bacterial Lo domains in vitro and in vivo. Studies will be extended to other pathogenic bacteria likely to contain Lo domains: Helicobacter pylori, the cause of ulcers and some cancers, and S. aureus, drug-resistant strains of which (MRSA) are a major public health threat. Studies will define the properties of bacterial domains, the principles behind their formation, and potential biomedical implications. Studies will then be extended to mammalian cells taking advantage of our discovery of a cyclodextrin that can fully exchange plasma membranes outer leaflet lipids with exogenous phospholipid and sphingolipid without disturbing cell sterols. Using this, how altering lipids modulate domain formation, properties, and function will be defined. This method will also be used to investigate plasma membrane lipid asymmetry and its function. Further development of the method will broadly impact biomembrane studies.

Public Health Relevance

New approaches are being developed and applied to define how, with the help of cholesterol or cholesterol-like molecules, the membranes surrounding cells, including membranes of pathogenic bacteria and mammalian cells, are organized into different regions (domains) with different molecular compositions. Understanding how membrane domains are organized should yield insights into new strategies to combat bacterial infections and human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
5R35GM122493-03
Application #
9665753
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Nie, Zhongzhen
Project Start
2017-04-01
Project End
2022-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
University-Wide
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Kim, JiHyun; Fukuto, Hana S; Brown, Deborah A et al. (2018) Effects of host cell sterol composition upon internalization of Yersinia pseudotuberculosis and clustered ?1 integrin. J Biol Chem 293:1466-1479
Zhang, Xiaoxue; London, Erwin; Raleigh, Daniel P (2018) Sterol Structure Strongly Modulates Membrane-Islet Amyloid Polypeptide Interactions. Biochemistry 57:1868-1879
Wang, Qing; London, Erwin (2018) Lipid Structure and Composition Control Consequences of Interleaflet Coupling in Asymmetric Vesicles. Biophys J 115:664-678
Toledo, Alvaro; Huang, Zhen; Coleman, James L et al. (2018) Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins. Mol Microbiol 108:63-76
Raj, Shriya; Nazemidashtarjandi, Saeed; Kim, Jihyun et al. (2017) Changes in glucosylceramide structure affect virulence and membrane biophysical properties of Cryptococcus neoformans. Biochim Biophys Acta Biomembr 1859:2224-2233
Kim, Ji Hyun; Singh, Ashutosh; Del Poeta, Maurizio et al. (2017) The effect of sterol structure upon clathrin-mediated and clathrin-independent endocytosis. J Cell Sci 130:2682-2695