scholarly journals Single molecule fluorescence for membrane proteins

Methods ◽  
2018 ◽  
Vol 147 ◽  
pp. 221-228 ◽  
Author(s):  
Oliver K. Castell ◽  
Patricia M. Dijkman ◽  
Daniel N. Wiseman ◽  
Alan D. Goddard
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Single Molecule Fluorescence

scholarly journals Single-molecule fluorescence vistas of how lipids regulate membrane proteins

2021 ◽  
Author(s):  
Alyssa E. Ward ◽  
Yujie Ye ◽  
Jennifer A. Schuster ◽  
Shushu Wei ◽  
Francisco N. Barrera
Keyword(s):  
Membrane Proteins ◽  
Physical Properties ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Short Review ◽  
Single Molecule Fluorescence ◽  
Determining Factors ◽  
Fluorescence Methods ◽  
Basic Understanding ◽  
Sample Heterogeneity

The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence methods can resolve sample heterogeneity, allowing to discriminate between the different molecular populations that biological systems often adopt. This short review highlights relevant examples of how SM fluorescence methodologies can illuminate the different ways in which lipids regulate the activity of membrane proteins. These studies are not limited to lipid molecules acting as ligands, but also consider how the physical properties of the bilayer can be determining factors on how membrane proteins function.

scholarly journals Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

2021 ◽  
Author(s):  
Vishal R Patel ◽  
Arturo M Salinas ◽  
Darong Qi ◽  
Shipra Gupta ◽  
David J Sidote ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Ion Channel ◽  
Single Molecule ◽  
Cyclic Nucleotide ◽  
Sensory Transduction ◽  
Single Molecule Fluorescence ◽  
Native Cell ◽  
Pore Opening ◽  
Intracellular Domains

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in nonnative lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. We further show that binding of the second ligand in the tetrameric channel is less cooperative than previously estimated from ensemble-averaged binding measures. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

scholarly journals Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vishal R. Patel ◽  
Arturo M. Salinas ◽  
Darong Qi ◽  
Shipra Gupta ◽  
David J. Sidote ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Ion Channel ◽  
Single Molecule ◽  
Cyclic Nucleotide ◽  
Sensory Transduction ◽  
Single Molecule Fluorescence ◽  
Native Cell ◽  
Pore Opening ◽  
Intracellular Domains

AbstractLigand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

scholarly journals An efficient GUI-based clustering software for simulation and Bayesian cluster analysis of single-molecule localization microscopy data

2021 ◽  
Author(s):  
Saskia Kutz ◽  
Ando C. Zehrer ◽  
Roman Svetlitckii ◽  
Gulce S. Gulculer Balta ◽  
Lucrezia Galli ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Membrane Proteins ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Cluster Algorithms ◽  
Bayesian Cluster Analysis ◽  
Microscopy Data ◽  
Bayesian Cluster ◽  
Single Molecule Fluorescence Microscopy

Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster's constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

scholarly journals An Efficient GUI-Based Clustering Software for Simulation and Bayesian Cluster Analysis of Single-Molecule Localization Microscopy Data

2021 ◽  
Vol 1 ◽  
Author(s):  
Saskia Kutz ◽  
Ando C. Zehrer ◽  
Roman Svetlitckii ◽  
Gülce S. Gülcüler Balta ◽  
Lucrezia Galli ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Membrane Proteins ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Cluster Algorithms ◽  
Bayesian Cluster Analysis ◽  
Microscopy Data ◽  
Bayesian Cluster ◽  
Single Molecule Fluorescence Microscopy

Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster’s constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

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