Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Anantharam, Arun; Onoa, Bibiana; Edwards, Robert H.; Holz, Ronald W.; Axelrod, Daniel

doi:10.1083/jcb.200908010

Cited by 120 publications

(166 citation statements)

References 46 publications

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“…Polarized total internal reflection (pTIRF) microscopy may be the best experimental technique where these analytical techniques could be used to great effect to simulate the expected polarization parameters of fluorophores in known domains [41,42]. In this technique, excitation and emission takes place in a thin layer near a surface and membrane events can be recorded with both spatial and temporal resolution [11,43]. A combination of molecular dynamics simulations of the fluorophores in the membranes and modeling of the spatial orientation of the evanescent wave using the presented approach would be a natural next step to validate and further explore the data obtained from this technique.…”

Section: Discussionmentioning

confidence: 99%

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From molecular dynamics to fluorescence anisotropy of fluorophores bound to oriented structures

Mazzeo

Busath

2013

Journal of Computational Physics

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Section: Discussionmentioning

confidence: 99%

“…Membrane conditions are of interest in cases of foraging, phototaxis, magnetotaxis, or other phenomena resulting in oriented single cells [4][5][6][7][8][9]. Embedded fluorophores may be useful to analyze bacterial behavior near surfaces [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

From molecular dynamics to fluorescence anisotropy of fluorophores bound to oriented structures

Mazzeo

Busath

2013

Journal of Computational Physics

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“…P/S varies with the relative intensities of the p-and s-pol excitations, biases in the optical system, and interference fringes. To reduce these effects, normalize the P/S ratios from obtained from diD emission to the ratio obtained with a solution containing 10 mM rhodamine 18 . 3.…”

Section: Image Analysismentioning

confidence: 99%

“…Pixel-to-pixel ratio images (P/S) reporting on the emission from sequential p-and s-pol excitation of diD will therefore specifically highlight regions of membrane deformation. In theory, the P/S ratio images are sensitive to even small angles of plasma membrane deviation from the coverglass, with the amplitude of the changes predicted by computer simulations 18 …”

Section: Introductionmentioning

confidence: 99%

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Imaging Plasma Membrane Deformations With pTIRFM

Passmore

Rao

Peleman

et al. 2014

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To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

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Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events

2012

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Total internal reflection fluorescence (TIRF) microscopy is a high‐contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z‐resolution of an epi‐fluorescence microscope. This technique also increases signal‐to‐noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through‐the‐objective TIRF illuminator and a prism‐based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through‐the‐objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi‐ and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed. Curr. Protoc. Cytom. 61:12.29.1‐12.29.19. © 2012 by John Wiley & Sons, Inc.

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Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Abstract: Imaging of individual secretory granules reveals how exocytosis curves the membrane.

Cited by 120 publications

References 46 publications

From molecular dynamics to fluorescence anisotropy of fluorophores bound to oriented structures

From molecular dynamics to fluorescence anisotropy of fluorophores bound to oriented structures

Imaging Plasma Membrane Deformations With pTIRFM

Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events

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