The long-term goals of this project are to develop methods to probe the organization and dynamic reorganization of lipids and proteins in biological membranes and to apply these methods to problems of broad biological importance. The lipid bilayer is the basic structure common to biological membranes. Membrane fluidity is critical for biological functions that depend upon conformational changes within membranes, the lateral association of lipids and proteins, their reorganization and pattern formation when cells interact, and processes that change membrane topology such as membrane fusion. This proposal describes model membrane architectures, along with imaging and analytical methods that probe these basic aspects of membrane dynamics. Three novel model membrane architectures have been developed and are essential for this work: (i) patterned supported bilayers, used for organizing and locating regions of interest for parallel imaging by mass spectrometry, super-resolution optical microscopy and atomic force microscopy;(ii) DNA-lipid tethered mobile vesicles whose individual collisions and interactions can be observed directly;and (iii) DNA-lipid tethered bilayer patches and giant vesicles, which serve as realistic substrates for membrane fusion and bilayer-bilayer interactions with control of curvature. Two problems of current biological and biomedical significance have been selected that exploit these architectures. (i) The mechanism of vesicle fusion orchestrated either by novel DNA-lipid conjugates or the natural neuronal protein fusion machinery. Tethered vesicles or bilayer patches will be used to precisely probe the steps of fusion at the single vesicle and single molecule level (Aim 1). We ultimately plan to assemble an artificial synapse in which each step and contribution of individual protein components and calcium can be assessed quantitatively. (ii) The organization of lipids and membrane proteins will be measured by using a new type of high spatial resolution and high sensitivity imaging mass spectrometry technique (Aim 2). Specific targets include the quantitative analysis of the composition of individual small vesicles, collective behavior of components believed to be associated in membrane rafts, and ultimately studies of lipid and protein clustering and reorganization in the immunological synapse.
Each Aim targets a significant area of membrane biophysics that integrates innovative molecular assemblies, interfacial fabrication, and advanced imaging methods to address a complex problem of wide interest.

Public Health Relevance

A significant fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. This proposal outlines new methods for studying membranes and membrane-associated proteins that can impact our understanding of biological function and organization, as well as impact biotechnology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM069630-09A1
Application #
8369852
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2004-01-01
Project End
2016-07-31
Budget Start
2012-09-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$309,798
Indirect Cost
$89,798
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Hughes, Laura D; Rawle, Robert J; Boxer, Steven G (2014) Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers. PLoS One 9:e87649
Hughes, Laura D; Boxer, Steven G (2013) DNA-based patterning of tethered membrane patches. Langmuir 29:12220-7
van Lengerich, Bettina; Rawle, Robert J; Bendix, Poul Martin et al. (2013) Individual vesicle fusion events mediated by lipid-anchored DNA. Biophys J 105:409-19
Lozano, Monica M; Liu, Zhao; Sunnick, Eva et al. (2013) Colocalization of the ganglioside G(M1) and cholesterol detected by secondary ion mass spectrometry. J Am Chem Soc 135:5620-30
Chung, Minsub; Koo, Bon Jun; Boxer, Steven G (2013) Formation and analysis of topographical domains between lipid membranes tethered by DNA hybrids of different lengths. Faraday Discuss 161:333-45; discussion 419-59
Rawle, Robert J; van Lengerich, Bettina; Chung, Minsub et al. (2011) Vesicle fusion observed by content transfer across a tethered lipid bilayer. Biophys J 101:L37-9
Levinson, Nicholas M; Bolte, Erin E; Miller, Carrie S et al. (2011) Phosphate vibrations probe local electric fields and hydration in biomolecules. J Am Chem Soc 133:13236-9
Chung, Minsub; Boxer, Steven G (2011) Stability of DNA-tethered lipid membranes with mobile tethers. Langmuir 27:5492-7
van Lengerich, Bettina; Rawle, Robert J; Boxer, Steven G (2010) Covalent attachment of lipid vesicles to a fluid-supported bilayer allows observation of DNA-mediated vesicle interactions. Langmuir 26:8666-72
Lozano, Monica M; Starkel, Cambrie D; Longo, Marjorie L (2010) Vesicles tethered to microbubbles by hybridized DNA oligonucleotides: flow cytometry analysis of this new drug delivery vehicle design. Langmuir 26:8517-24

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