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Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

ABSTRACT: The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, ?-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, ?2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.

SUBMITTER: Latty SL

PROVIDER: S-EPMC4643199 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

Latty Sarah L SL Felce James H JH Weimann Laura L Lee Steven F SF Davis Simon J SJ Klenerman David D

Biophysical journal 20151101 9

The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respec ...[more]

PMID: 26536257

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Project description:A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

Dataset Information

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

Publications

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

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