It has very recently been recognized that numerous proteins involved in membrane curvature (MC) sensing and regulation are also implicated in a variety of diseases. Thus, the need to understand how MC couples with cellular function has emerged as a focus area both from a basic research as well as a biomedical perspective. Several membrane-binding protein domains have been shown to be capable of generating MC. Some of the most prominent members of these protein domains are the Bin/amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH) domains. These domains have been found in numerous disease-related proteins. To date, studies of these proteins rely heavily on electron microscopy imaging. The mechanisms by which these domains couple to MC and in particular a quantitative understanding of the hypothesized mechanisms have remained elusive. Membrane curvature sensing and generating proteins can be distinguished by several different types of disease involvement mechanisms. Firstly, altered expression levels are often found in malignant cells. For example, down-regulation or mis-splicing of BAR domain-containing proteins is found in cancers of various organs. Secondly, specific point mutations are encountered in proteins related to MC. For example, several point mutations in the BAR domain of the protein BIN1 (amphiphysin-2) are found to be linked to centronuclear myopathy. These mutations have been connected with membrane tubulation (i.e. curvature generation) activity. Furthermore, mutations within SH3 domains of BAR proteins, or mutations in SH3 domain ligands, are linked to several diseases. Our goal is to understand how protein concentration affects MC sensing and MC generation, and by which mechanisms these phenomena are effected by wild type and strategically mutated proteins. We will also investigate how ligand/SH3 domain interactions couple to membrane curvature. In order to achieve this goal, we have developed a biophysical approach that allows us to separately quantify MC sensing and generation. Curvature sensing will be determined through fluorescence measurements. These will monitor curvature-dependent re-localization of membrane-binding proteins and their mutants on membranes with precisely adjustable curvature. MC generation will be quantified through probing mechanical aspects of bent membranes. We will focus on using micropipette-aspiration and optical trapping to manipulate cylindrical membranes pulled from vesicles that are either self-assembled or purified from cells. We will provide improved mechanistic understanding of our quantitative data by comparing them to statistical mechanical, thermodynamic and mechanical theories. Our studies will provide valuable insight into MC regulation and sensing by proteins which may aid in understanding the role that these proteins play in numerous diseases.

Public Health Relevance

Membrane proteins coupling with cellular membrane curvature are involved in numerous diseases, including Alzheimer's, Huntington's, and Parkinson's disease, arthritis, centronuclear myopathy, diabetes, epilepsy, mental retardation, stiff person syndrome, and several types of cancer. The field of experimental research into curvature regulation and sensing by peripheral proteins is currently dominated by qualitative observations and insufficient mechanistic understanding. This project aims at providing a quantitative understanding of membrane curvature sensing and generation of three important classes of proteins, including mutants that occur in diseases such as centronuclear myopathy and Parkinson's disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM097552-01A1
Application #
8194640
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2011-09-01
Project End
2016-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
1
Fiscal Year
2011
Total Cost
$286,384
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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