Over the past half century, as molecular biology and biochemistry have developed to inform our understanding of disease, and as this understanding has driven the search for treatments in biotechnology and molecular medicine, the dominant theoretical scaffold for interpreting molecular behavior has been the structure-function relationship. Our understanding of molecular biology's structure-function relationships is largely limited, however, to the molecules of the central dogma: proteins and oligonucleotides. The vast variety of molecular structure outside of the central dogma, particularly among lipids and carbohydrates, suggests that these, too, can be understood in terms of structure driving function. In particular, there has been intense interest in the functional role that lipids might play in the plasma membrane. This project deploys a new class of synthetic lipid bilayers to begin drawing connections between the structure of lipid molecules-both in terms of individual molecular structure and supermolecular organization-and the function of the plasma membrane. The research tools developed and deployed here, called asymmetric giant unilamellar vesicles (AGUVs), are designed to uniquely mimic the cell membrane, capturing properties such as compositional asymmetry and molecular crowding better than other existing synthetic lipid bilayers. One of the many questions that AGUVs can help answer involves passive transport across the cell membrane. Passive transport is an important route for drug delivery and passage of environmental toxins into cells. Recent results show that the mechanism of this transport is complex, and highly dependent on lipid behavior. This project deploys an AGUV-based technique for systematically measuring the dynamics of solute molecules interacting with and penetrating lipid bilayers, yielding richer mechanistic data than other approaches are capable of delivering. AGUVs can also be used to study the mechanical properties of the cell membrane. These properties- particularly resistance to bending-control protein function and are important in a range of physiological processes. While synthetic lipid bilayers have been used to probe these properties, little is known about the effects of bilayer asymmetry on them. This project uses AGUVs to discover these effects. Lipid interactions with integral membrane proteins are likely a major mode by which lipids influence cell behavior. Very little is known, however, about the origins or controlling parameters of these interactions. This project begins to untangle this problem in AGUVs by using fluorescence microscopy to probe how peptides modeling the transmembrane regions of various proteins associate with segregated lipid domains. Finally, AGUVs can facilitate the systematic study of the effects of macromolecular crowding in the cell interior. The microfluidic technique by which AGUVs are formed allows for the inclusion of arbitrary molecules within them, leading to unique molecularly crowded structures.

Public Health Relevance

Cells are surrounded by membranes that consist of two layers of lipid molecules, and the chemical composition is different in these two layers. In this project, a new type of artificial cell membrane that is uniquely capable of mimicking this asymmetry is deployed to study a range of important biological processes, including drug transport into cells, mechanical deformation of the cell membrane, and interactions between lipid molecules and receptor proteins involved in cancer treatment and diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093279-04
Application #
8534183
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2010-09-30
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$281,543
Indirect Cost
$100,026
Name
University of Southern California
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Bhargava, Krisna C; Thompson, Bryant; Malmstadt, Noah (2014) Discrete elements for 3D microfluidics. Proc Natl Acad Sci U S A 111:15013-8
Gutierrez, M Gertrude; Malmstadt, Noah (2014) Human serotonin receptor 5-HT(1A) preferentially segregates to the liquid disordered phase in synthetic lipid bilayers. J Am Chem Soc 136:13530-3
Cruz, Jonathan W; Rothenbacher, Francesca P; Maehigashi, Tatsuya et al. (2014) Doc toxin is a kinase that inactivates elongation factor Tu. J Biol Chem 289:7788-98
López Mora, Néstor; Hansen, Jesper S; Gao, Yue et al. (2014) Preparation of size tunable giant vesicles from cross-linked dextran(ethylene glycol) hydrogels. Chem Commun (Camb) 50:1953-5
Dunkle, Jack A; Vinal, Kellie; Desai, Pooja M et al. (2014) Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA. Proc Natl Acad Sci U S A 111:6275-80
Solmaz, Mehmet E; Sankhagowit, Shalene; Biswas, Roshni et al. (2013) Optical stretching as a tool to investigate the mechanical properties of lipid bilayers. RSC Adv 3:
Dayani, Yasaman; Malmstadt, Noah (2013) Liposomes with double-stranded DNA anchoring the bilayer to a hydrogel core. Biomacromolecules 14:3380-5
Fagan, Crystal E; Dunkle, Jack A; Maehigashi, Tatsuya et al. (2013) Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state. Proc Natl Acad Sci U S A 110:9716-21
Hansen, Jesper S; Thompson, James R; Helix-Nielsen, Claus et al. (2013) Lipid directed intrinsic membrane protein segregation. J Am Chem Soc 135:17294-7
Hu, Peichi C; Li, Su; Malmstadt, Noah (2011) Microfluidic fabrication of asymmetric giant lipid vesicles. ACS Appl Mater Interfaces 3:1434-40

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