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Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy.

ABSTRACT: Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C(20) or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions.

SUBMITTER: Oreopoulos J

PROVIDER: S-EPMC2717349 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy.

Oreopoulos John J Yip Christopher M CM

Biophysical journal 20090301 5

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was de ...[more]

PMID: 19254557

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Project description:The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

Dataset Information

Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy.

Publications

Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy.

Similar Datasets

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