Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Tobias Baumgart

Second Advisor

Ivan Dmochowski

Abstract

The cellular membrane is a highly heterogeneous and dynamic system whose compositions and morphologies are delicately controlled by the interplay between lipids and proteins. For instance, membranes with distinct curvature exist in various types of organelles. Dynamic membrane remodeling is essential in a variety of physiological functions. Membrane remodeling serves as an active regulatory mechanism in eukaryotic cells to locally accumulate lipids or proteins as hot spots to transduce signals. Membrane reshaping processes are found in important cellular activities, such as cell migration, division, viral budding/fusion, and endocytosis. Bin/Amphiphysin/Rvs (BAR) domain proteins are a group of peripheral proteins capable of sensing, generating and stabilizing curved membranes. Mutations or deletions of BAR domains impair membrane tubulation inside cells and often lead to onset of human diseases. In this dissertation, I aim to provide biophysical measurements to understand the molecular mechanisms by which BAR domain proteins reshape the underlying membranes and how membrane curvature generation is regulated. Furthermore, this study aims to shed light on how disease- associated mutations in BAR domains contribute to the pathogenesis of human myopathies.

The protein I am interested in is called BIN1 isoform8, an N-BAR domain containing protein exclusively expressed in striated muscle tissues. BIN1 functions in the biogenesis of T-tubules, which are invaginations from the plasma membrane. They propagate action potentials to accomplish excitation-contraction coupling. Three mutations (K35N, D151N and R154Q) in the BAR domain of BIN1 have been discovered in patients with centronuclear myopathy (CNM), a congenital muscular disorder in which nuclei are centrally mislocated, and impaired organization of T-tubules has been reported. I found that BIN1 disease mutants disrupt membrane tubulation both in vivo and in vitro. The deformation capacity of BIN1 N-BAR mutants strongly depends on lipid composition. The R154Q mutant generates smaller membrane curvature compared to wild-type (WT) N-BAR, while the D151N mutant is unable to tubulate membranes under certain experimental conditions. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appears to be the primary reason for the observed deformation incapacity. Interestingly, none of the mutants showed a significantly compromised curvature sensing ability.

At modest protein concentrations, `budding' structures were found on liposomes that are hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutant is unable to oligomerize on membranes. Although an insignificant difference between WT and K35N N-BAR was found in in vitro assays, actin depolymerization in live cells allowed tubulation of plasma membranes through the K35N mutant.

Another focus of this dissertation is to understand how the membrane curvature sensing & generation (MC-S&G) ability of BIN1 is regulated in the full-length (FL) BIN1. In addition to the N-terminal BAR domain, BIN1 contains a muscle-specific polybasic motif (exon10) as the phosphoinositide binding module and a C-terminal Src Homology 3 (SH3) domain for the downstream signaling protein recruitments, such as dynamin2 in muscle cells. We hypothesize that the interactions between exon10 and SH3 domain allow an autoinhibitory regulation in BIN1-FL. We established that the SH3 domain binds to the exon10 motif stronger than the proline-rich domain (PRD) of dynamin2 due to the electrostatic nature of exon10-SH3 interactions. In addition to blocking dynamin2 membrane recruitment by the exon10-SH3 complex, we found that the MC-S&G ability of BIN1-FL is inhibited on membranes lacking PI(4,5)P2. Addition of PI(4,5)P2 in the membranes activates BIN1 to sense and induce membrane curvature. The co-presence of SH3 domain and exon10 motif leads to the strongest phosphoinositide-mediated control of BIN1 function. Addition of SH3 domain ligand (such as dynamin2 derived PRD peptide), as well as addition of water-soluble PI(4,5)P2 analog, can both enhance the MC-S&G ability of BIN1 on PI(4,5)P2-absent membranes, indicating that the key step to activate BIN1 is to disrupt the exon10-SH3 interaction. Nonsense mutation K436X, found in centronuclear myopathy (CNM) patients, abolishes the SH3 domain binding with both exon10 and PRD motif, resulting in increased membrane deformation capacity. Overall, our results suggest an autoinhibition model for BIN1 that involves a synergistic regulation by membrane composition and protein- protein interactions.

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