Vesicular membrane fusion involves the release of contents in a broad array of biological systems, such as intracellular trafficking, secretion, fertilization, and development. It is also a critical step in the infection of cells by membrane enveloped viruses such as HIV, influenza, and Ebola. SNARE proteins form the core of the fusion machinery in nearly all intracellular fusion processes. The initial complete connection between two fusing membranes is the fusion pore. There is considerable evidence that both the fusion machinery and the biophysical properties of the membranes themselves affect contents release, lipid mixing, and fusion kinetics, but the mechanisms are poorly understood. Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single pore dynamics the contributions from different influences are almost impossible to separate. This thesis examines the biophysical mechanisms by which SNAREs and lipid composition control fusion rates and fusion pore kinetics.
First, we studied fusion pore flickering in vitro. We used total internal reflection fluorescence (TIRF) microscopy to quantify fusion pore dynamics in vitro and to separate the roles of SNARE proteins and lipid bilayer properties. To interpret the experimental measurements quantitatively, we developed a mathematical model to describe the diffusion of labelled lipids from a vesicle, through a flickering fusion pore, and into a supported bilayer. When small unilamellar vesicles (SUV) bearing neuronal v SNAREs fused with planar bilayers (SBL) reconstituted with cognate t SNARES, lipid transfer rates were severely reduced, suggesting that pores flickered. We developed an algorithm which included a complete description of fluorophores in the TIRF field. We accounted for the intensity decay of the evanescent TIRF wave normal to the SBL, the polarization of the evanescent TIRF wave, and any potential quenching effects. In general, the first two effects are coupled. This algorithm allowed us to measure the sizes of docked vesicles using fluorescent microscopy.
From the lipid release times we used the model to compute pore openness, the fraction of the time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested SNARE mediated and non specifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t SNAREs to the fusion site, consistent with t SNAREs being pre clustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion pore bending energy, and indirectly by concentrating a number of SNAREs into individual fusion events.
In the second part of the thesis, I describe my contributions to a project in which a mathematical model was developed to describe the behavior of SNAREpins connecting SUVs of different sizes to a planar membrane. It was necessary to quantify the membrane membrane and SNAREpin membrane interaction forces. By combining the well known van der Waals, electrostatic, and steric hydration membrane forces with the SNAREpin membrane electrostatic interactions I developed a complete description of the membrane forces involved in SUV-SBL fusion. We then combined the description of the interactions with experimentally measured SNARE zippering energies. We find that the predominant driving forces for membrane fusion, once the SNAREpins have completely zippered, are steric hydration forces among the SNAREpins and membranes. These forces enlarge a SNAREpin cluster, which in turns pulls the membranes together due to curvature effects.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8G15ZM8 |
| Date | January 2015 |
| Creators | Stratton, Benjamin Somerall |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
Page generated in 0.0047 seconds