Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Cutrale, Francesco; Rodríguez, Daniel; Hortigüela, Verónica; Chiu, Charles B.; Otterstrom, Jason; Mieruszynski, Stephen; Seriola, Anna; Larrañaga, Enara; Raya, Ángel; Lakadamyali, Melike; Fraser, Scott E.; Martínez, Elena; Ojosnegros, Samuel

doi:10.1038/s41596-018-0111-9

Cited by 41 publications

(35 citation statements)

References 95 publications

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“…Although SpIDA has many useful attributes (21), as for related methods including Number and Brightness analysis (29) and Photon Counting Histogram (30), it only provides information on the average size of oligomers within potentially complex mixtures of oligomers of different sizes and also may be sensitive to brighter inhomogeneities in the fluorescence images. To overcome these issues we recently developed a new approach described as 1-and 2-dimensional Fluorescence Intensity Fluctuation (FIF) spectrometry (22,23).…”

Section: Fluorescence Intensity Fluctuation (Fif) Spectrometry Reinfomentioning

confidence: 99%

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

Ward

Pediani

Marsango

et al. 2021

Journal of Biological Chemistry

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CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested the receptor may exist as a dimer or oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means we confirm that CXCR4 can exist as a mixture of monomers, dimers and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t— a small, drug-like isothiourea derivative — rapidly destabilizes the oligomeric structure, while AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

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Section: Fluorescence Intensity Fluctuation (Fif) Spectrometry Reinfomentioning

confidence: 99%

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

Ward

Pediani

Marsango

et al. 2021

Journal of Biological Chemistry

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“…The fluctuation intensity ( F ( t )) was further extracted through photon counting histogram analysis (Figure S4D‐F). Compared with the effect of laser pulse energy on the intensity of R6G fluorescence, the TPL intensity of AuNR and AuNR‐RB were enhanced as the laser pulse energy increased. And then, the average number of emissive species (1/˂ N ˃) yielded from the amplitude of autocorrelation function also suggested that the number of emissive species in R6G solution was not affected by the laser pulse energy (Figure S4G).…”

Section: Resultsmentioning

confidence: 99%

Visualization photofragmentation‐induced rhodamine B release from gold nanorod delivery system by combination two‐photon luminescence imaging with correlation spectroscopy

Dong

Chen

Yang

et al. 2020

Journal of Biophotonics

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Plasmon-enhanced gold nanorod (AuNR) with high photothermal conversion efficiency is a promising lightcontrollable nanodrug delivery system for cancer therapy. Understanding the mechanism for the light-controllable drug release of AuNR delivery systems is important for the development of nanomedicine. In this study, the rhodamine B (RB) released from AuNR-RB nanodelivery system was quantitated and visualized by using twophoton luminescence (TPL) imaging combined with correlation spectroscopy.The photofragmentation of AuNR induced by femtosecond pulsed laser was revealed by TPL correlation spectroscopy when the laser energy was above the thermal damage threshold of AuNR, and the RB released from this nanodrug delivery system was visualized by TPL imaging. Furthermore, the photofragmentationinduced release of RB from AuNR-RB nanodelivery system was visualized in liv- ing MCF-7 breast cancer cells by TPL imaging combined with correlationAbbreviations: AuNR, gold nanorod; RB, rhodamine B; R6G, rhodamine 6G; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000PEG]; AuNR-RB, gold nanorod-RB nanodelivery system; DSPE@AuNR-RB, DSPE encapsulated AuNR-RB; FCS, fluorescence correlation spectroscopy; TEM, transmission electron microscope; DLS, dynamic light scattering; FRET, Förster resonance energy transfer.Shiqing Dong and Xiuqin Chen contributed equally to this study.

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“…Hortiguela et al developed a statistical resampling of raw fluctuation data to measure coexisting oligomer species in each pixel and this improved N&B methodology is called enhanced N&B analysis (eN&B) [154,155]. One drawback to eN&B is analysis requires 200 frames and can take from seconds to minutes to complete one timepoint.…”

Section: Number and Brightness (Nandb) Analysismentioning

confidence: 99%

“…sliding window, and the exponential filtering method [143]. Filtering algorithms were applied to imaging FFT data to remove contributions from cellular movement or photobleaching and thus enhance the accuracy of the calculated molecular parameters [140,143,155,173]. Lange et al used a wavelet shrinking algorithm to reduce the false positive signal from 69% to 4% for the negative control in FCCS measurements [174].…”

Section: Advantages and Disadvantages Of Fftsmentioning

confidence: 99%

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

Youker¹,

Voet²

2020

Fluorescence Methods for Investigation of Living Cells and Microorganisms

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G-protein-coupled receptors (GPCRs) are seven transmembrane receptors that form the largest superfamily of signaling proteins, and the family members function in a diverse array of metabolic pathways including cardiac function, immune response, neurotransmission, smell, taste, cell differentiation and growth, and vision. It is becoming clear that alteration in the quaternary structure of the GPCR receptor can have a profound impact on signaling capabilities. Biochemical, biophysical, physiological, x-ray crystallographic, and computational methods have been used over the last 40-50 years to study the structure and function of GPCRs. Evidence from these studies confirm that GPCRs can be organized as monomers, dimers, and higher-order oligomers. However, many times, these methods require extraction of the receptor from its native environment and high levels of expression and only provide a snapshot of information. A need arose for techniques that could measure the assembly and disassembly of receptors at few-to-single molecule resolution in their native environment at fast time scales. In the last 20 years, fluorescence fluctuation techniques have filled this need and provided new insight into the dynamics of GPCR organization in the absence and presence of ligands, agonists, and antagonists. In this book chapter, we provide a brief introduction to GPCR structure and function [Section 1]. An overview of the theoretical basis for fluorescence fluctuations techniques (FFTs) and how FFTs can be used to study the oligomeric structure of GPCRs in live and fixed cells is explained [Section 2]. We discuss the advantages and limitations of FFTs [Section 3], and finally, we summarize select case studies on GPCR structure and function revealed by FFT experiments [Section 4].

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Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Cited by 41 publications

References 95 publications

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

Visualization photofragmentation‐induced rhodamine B release from gold nanorod delivery system by combination two‐photon luminescence imaging with correlation spectroscopy

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

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