A high resolution electro-optical approach for investigating transition of soluble proteins to integral membrane proteins probed by colicin A

Honigmann, Alf; Pulagam, Lakshmi Padmavathi; Sippach, Michael; Bartsch, Philipp; Steinhoff, Heinz‐Jürgen; Wagner, Richard

doi:10.1016/j.bbrc.2012.09.069

Cited by 5 publications

(4 citation statements)

References 26 publications

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“…These interactions are likely modulated by the two main membrane ultra-structure elements[ 1 – 7 ]. Some diffusing proteins are corralled between “fences” created by cytoskeleton-anchored membrane-associated proteins[ 8 ]; other diffusing proteins are transiently captured or trapped in either protein nanoclusters or cholesterol-dependent lipid nanodomains, so-called lipid rafts[ 2 , 3 , 9 ]. Both structures are too small and too dynamic to be directly imaged by optical microscopy.…”

Section: Introductionmentioning

confidence: 99%

Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

et al. 2015

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Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein’s diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein’s tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.

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Section: Introductionmentioning

confidence: 99%

Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

et al. 2015

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“…We first briefly summarize and emphasize basic critical topics for setting up such a system. Similar combined or simultaneous optical recordings on channel proteins have been performed previously in artificial bilayers [3,4], giant liposomes [5,6] oocytes [7,8,9] and the droplet interface [10]. Further on, we describe a combination of the above two approaches using cushions of natural or synthetic hydrogels as bilayer support which up to now have not been used frequently, despite offering significant advantages in many aspects [11,12,13,14].…”

Section: Introductionmentioning

confidence: 88%

Horizontal Bilayer for Electrical and Optical Recordings

et al. 2012

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Artificial bilayer containing reconstituted ion channels, transporters and pumps serve as a well-defined model system for electrophysiological investigations of membrane protein structure–function relationship. Appropriately constructed microchips containing horizontally oriented bilayers with easy solution access to both sides provide, in addition, the possibility to investigate these model bilayer membranes and the membrane proteins therein with high resolution fluorescence techniques up to the single-molecule level. Here, we describe a bilayer microchip system in which long-term stable horizontal free-standing and hydrogel-supported bilayers can be formed and demonstrate its prospects particularly for single-molecule fluorescence spectroscopy and high resolution fluorescence microscopy in probing the physicochemical properties like phase behavior of the bilayer-forming lipids, as well as in functional studies of membrane proteins.

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“…Principal setup of a horizontal lipid bilayer (HLB) combined with a confocal scanning spectrometer allowing high resolution fluorescence imaging and spectroscopy [132–134]. …”

Section: Electrophysiology On Lipid Bilayers Combined With Fluorescenmentioning

confidence: 99%

“…One attempt to follow membrane transport of uncharged solutes at a single molecule level in HLBs, is the optical single transporter recording approach, using confocal microscopy and HLB-arrays [138]. The combination of highresolution fluorescence and electrical recording allows the monitoring of the transition of water soluble toxins, which form high conducting membrane pores upon transition from solution into the membrane [132]. Corre spondingly, it should be possible to monitor the transition of water soluble receptor-cargo complexes into the membrane, thereby forming membrane pores [139].…”

Section: Electrophysiology On Lipid Bilayers Combined With Fluorescen...mentioning

confidence: 99%

The 2018 correlative microscopy techniques roadmap

Ando

Bhamidimarri

Brending³

et al. 2018

J. Phys. D: Appl. Phys.

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110

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Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell–cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure–function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

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A high resolution electro-optical approach for investigating transition of soluble proteins to integral membrane proteins probed by colicin A

Cited by 5 publications

References 26 publications

Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

Horizontal Bilayer for Electrical and Optical Recordings

The 2018 correlative microscopy techniques roadmap

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