Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.02.07.527542

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells

Petrich, A.·Aji, A. K.·Dunsing, V.·Chiantia, S.
View original source ↗Export metadata ↓

Abstract

Protein-protein-interactions play an important role in several cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for Protein-protein-interactions quantification via fluorescence fluctuation-based methods.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Petrich, A., Aji, A. K., Dunsing, V., Chiantia, S.. 2023-02-07. Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells. https://doi.org/10.1101/2023.02.07.527542

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Lipid Scrambling Amplifies Plasma Membrane Undulations via Anti-Registered Domains Stabilized by Long-Chain Sphingomyelin

Lipid scrambling, which dissipates the transbilayer lipid asymmetry of plasma membranes (PMs), plays an essential role in various large-scale membrane remodeling processes. Although lipid domains have been implicated in these processes, the underlying mechanisms remain poorly understood, owing to the lack of a molecular-level picture of how lipid scrambling reorganizes these domains. Here, using coarse-grained molecular dynamics simulations of asymmetric and fully scrambled PM models, we show that lipid scrambling markedly enhances membrane undulations. We identify two key features of the domain organization underlying this enhancement in the scrambled PM: domain anti-registration, which couples to membrane curvature through a mismatch in preferred curvature between the two leaflets, and scrambling-induced domain enlargement, which permits this curvature to extend over large length scales. Simulations with different sphingomyelin (SM) compositions demonstrate that long-chain SMs stabilize anti-registered domains and the associated membrane curvature. Together, these results suggest a physical mechanism by which lipid scrambling primes the PM for remodeling through the reorganization of lipid domains, with long-chain SMs playing a key role. We also find that the interleaflet domain organization of the scrambled PM model depends strongly on system size, shifting from anti-registration to registration as the simulation box is reduced, and we discuss the origin of this finite-size effect.

biophysics↗
bioRxiv2026

Evaluating speech-like distortion product otoacoustic emissions: a comparison of time- and frequency-domain metrics across three datasets

Speech-like distortion-product otoacoustic emissions (DPOAEs) allow cochlear activity to be measured during the perception of natural, continuous speech and have been used to study attentional effects in the cochlea. The technique is still young, however, and existing studies differ in stimulus design and in the metric used to quantify the emissions, which makes their results difficult to compare. Here, we reanalysed three existing datasets (106 participants, 14 stimulus types) with a common pipeline. We compared six metrics: the band power of the recording, the magnitude-squared coherence (MSC) between the recording and a DPOAE template, and four metrics derived from their complex cross-correlation, namely peak magnitude, signal-to-noise ratio (SNR), peak area and peak width. A separability index quantified how well each metric separated in-ear recordings from out-of-ear control recordings without emissions. Band power, MSC and SNR separated best, with mean separability indices of about 0.9, whereas the peak magnitude performed worst and was inverted for half of the stimulus types. Since band power does not require a DPOAE template, it serves as a sanity check of the setup rather than as evidence for an emission. The MSC was insensitive to the latency of the emission but well suited for comparisons across stimuli, whereas the cross-correlation additionally provided the latency. The emissions differed significantly between the stimulus types of the three datasets, and those below about 700 Hz were measured least reliably. We derive practical recommendations for the analysis and stimulus design of future experiments.

biophysics↗
bioRxiv2026

Overlap of competing voices may mask attentional effects on speech-like distortion product otoacoustic emissions

Whether selective auditory attention modulates the cochlear amplifier through the medial olivocochlear system remains debated. Using speech-like distortion-product otoacoustic emissions (DPOAEs), elicited by waveforms that follow the harmonics of natural speech, we previously found that selectively attending one of two competing talkers reduced the emissions related to the resolved harmonics of the female voice. Here, we attempted to reproduce this effect in a design that separated different frequency regimes and harmonic resolvability. Forty-four participants attended one of two competing talkers, a female and a male voice, presented to one ear, while speech-like DPOAEs related to both voices were recorded from the other ear. Stimuli were derived from resolved and unresolved harmonics in a low-frequency (up to 1.5 kHz) and a transitional (1.5 to 4 kHz) regime, which required pitch-shifting both voices so that their fundamental frequencies were much closer to each other than in the original study. Speech-like DPOAEs emerged for most stimulus types, although they were weak for some and absent for one. Their amplitude, however, did not depend on attention for any stimulus type. Further time-domain metrics showed only isolated attentional effects of opposite direction for two stimulus types, and a frequency-domain metric showed none. A cochlear excitation model suggests that, due to the closely spaced fundamental frequencies of the two voices, the emissions most comparable to the original study presumably arose at cochlear locations shared by both voices, where attention would modulate both voices alike.

biophysics↗