scholarly journals Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

2021 ◽  
Author(s):  
Kristina Cechova ◽  
Chenyang Lan ◽  
Matus Macik ◽  
Nicolas P. F. Barthes ◽  
Manfred Jung ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Single Molecule ◽  
Protein Interactions ◽  
Live Cell Imaging ◽  
Specific Effect ◽  
Single Molecule Imaging ◽  
Physiological Conditions ◽  
Biochemical Assays ◽  
A Cell ◽  
Membrane Density

AbstractOpioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

scholarly journals Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

2020 ◽  
Author(s):  
Kristina Cechova ◽  
Chenyang Lan ◽  
Nicolas P. F. Barthes ◽  
Manfred Jung ◽  
Maximilian H. Ulbrich
Keyword(s):  
Opioid Receptors ◽  
Single Molecule ◽  
Protein Interactions ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Specific Effect ◽  
Single Molecule Imaging ◽  
Mu Opioid Receptors ◽  
Mu Opioid ◽  
Physiological Conditions

AbstractOpioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions. Here we use three live-cell imaging approaches to assess dimerization of ORs at different expression levels. At high membrane densities, a split GFP assay reveals that κOR dimerizes, while μOR and δOR stay monomeric. In contrast, single-molecule imaging showed no κOR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities 100x higher than conventional single-molecule imaging. We observe that κOR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, μOR and δOR stay monomeric even at the highest densities covered. The observation of long-lasting κOR dimers but not higher order aggregates suggests that OR dimerization is a specific effect and not a result of increasing expression.

scholarly journals Dynamic instability of clathrin assembly provides proofreading control for endocytosis

2019 ◽  
Vol 218 (10) ◽  
pp. 3200-3211 ◽  
Author(s):  
Yan Chen ◽  
Jeffery Yong ◽  
Antonio Martínez-Sánchez ◽  
Yang Yang ◽  
Yumei Wu ◽  
...  
Keyword(s):  
Quality Control ◽  
Single Molecule ◽  
Dynamic Instability ◽  
Single Molecule Imaging ◽  
Coated Pits ◽  
A Cell ◽  
The Cost ◽  
Dynamic Exchange ◽  
Cargo Sorting

Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report that clathrin coat formation and cargo sorting can be uncoupled, indicating that a checkpoint is required for functional cargo incorporation. We demonstrate that the ATPase Hsc70 and a dynamic exchange of clathrin during assembly are required for this checkpoint. In the absence of Hsc70 function, clathrin assembles into pits but fails to enrich cargo. Using single-molecule imaging, we further show that uncoating takes place throughout the lifetime of the growing clathrin-coated pits. Our results suggest that the dynamic exchange of clathrin, at the cost of the reduced overall assembly rates, primarily serves as a proofreading mechanism for quality control of endocytosis.

scholarly journals Self-organization of parS centromeres by the ParB CTP hydrolase

Science ◽  
2019 ◽  
Vol 366 (6469) ◽  
pp. 1129-1133 ◽  
Author(s):  
Young-Min Soh ◽  
Iain Finley Davidson ◽  
Stefano Zamuner ◽  
Jérôme Basquin ◽  
Florian Patrick Bock ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Molecule ◽  
Dna Sequences ◽  
Self Organization ◽  
Single Molecule Imaging ◽  
Catalytic Center ◽  
Biochemical Assays ◽  
Promising Target ◽  
Cytidine Diphosphate ◽  
Cocrystal Structure

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS. ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

Nanoscale Analysis of the Effect of Pathogenic Mutations on Polycystin-1

2010 ◽  
Author(s):  
Liang Ma ◽  
Meixiang Xu ◽  
Andres F. Oberhauser
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Structure And Function ◽  
Eukaryotic Cells ◽  
Mechanical Forces ◽  
Force Microscopy ◽  
Physiological Conditions ◽  
Atomic Force ◽  
And Function

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells is the result of cycles of mechano-sensing, mechano-transduction, and mechano-response. Recently developed single-molecule atomic force microscopy (AFM) techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins, such as titin (an elastic mechano-sensing protein found in muscle) and polycystin-1 (PC1, a mechanosensor found in the kidney).

Single-stranded DNA loops as fiducial markers for exploring DNA–protein interactions in single molecule imaging

Methods ◽  
2013 ◽  
Vol 60 (2) ◽  
pp. 122-130 ◽  
Author(s):  
Oliver Chammas ◽  
Daniel J. Billingsley ◽  
William A. Bonass ◽  
Neil H. Thomson
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Single Molecule Imaging ◽  
Fiducial Markers ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
Dna Protein Interactions

scholarly journals Co-transcriptional quantification of RNA synthesis, RNA folding and RNA-protein interactions by "Systems NMR" approach.

2019 ◽  
Author(s):  
Yaroslav Nikolaev ◽  
Nina Ripin ◽  
Martin Soste ◽  
Paola Picotti ◽  
Dagmar Iber ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Folding ◽  
Single Sample ◽  
Rna Transcription ◽  
Biochemical Assays ◽  
Experimental Approaches ◽  
Reaction Constants ◽  
Time Systems

Abstract The protocol describes how to setup and analyse observation of a co-transcriptional RNA folding network by Systems NMR approach. While most experimental approaches can monitor only a single molecule class or reaction type at a time, Systems NMR permits single-sample dynamic quantification of entire “heterotypic” networks – involving different reaction and molecule types. It thus provides a deeper systems-level understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of “omics”. This particular protocol describes the reconstruction of an 8-reaction co-transcriptional network - with simultaneous monitoring of RNA, metabolite, and proteins in a single sample at the same time. From reactions side, the protocol simultaneously quantifies RNA transcription, RNA folding and RNA-protein interactions (observed both from RNA and from protein side) and few other auxiliary reactions. In addition to fundamental analyses of reaction constants under different conditions, the current applications of this particular reconstruction are: (1) map RNA-binding interfaces on proteins without having to purify/order the RNA; (2) monitor co-transcriptional RNA folding perturbations by proteins and small molecules; (3) monitor RNA-transcription-driven protein phase-separation with the possibility to observe multiple proteins at once, each with residue-level resolution. Not counting the protein and RNA template preparation times, the NMR measurement and data analysis parts take about 1 day each. This protocol accompanies Nikolaev et al, Nature Methods, 2019 (doi:XXX). The most up-to-date version of the protocol (including example code and data) is available at: github.com/systemsnmr/ivtnmr

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