A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Finan, Kieran; Raulf, Anika; Heilemann, Mike

doi:10.1002/ange.201505664

Cited by 4 publications

(7 citation statements)

References 20 publications

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“…Previous approaches have relied on calibrating blinking and other photophysical properties of fluorophores [42][43][44] , which cannot account for long-lived dark states and incomplete labeling or maturation. Furthermore, a variety of counting references have been developed including selfassembling oligomers 45 , DNA-structures 34 , receptors 32 , or a combination of monomers, dimers and trimers of mEos2 33,46 . While this is a powerful approach, a major limitation is the need for faithful segmentation, which is not trivial and often strongly dependent on algorithmic parameters.…”

Section: Counting Of Proteinsmentioning

confidence: 99%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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Section: Counting Of Proteinsmentioning

confidence: 99%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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“…Furthermore, to extend our investigation to larger protein complexes, we performed N&B measurements on U2OS cells expressing the dodecameric E.coli glutamine synthetase (GlnA) 32 . We measured an average normalized brightness of e12-mer=8.8 ± 0.3.…”

Section: Figure 1 Brightness Comparison Of Different Fps In Living Hmentioning

confidence: 99%

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Dunsing

Luckner

Zühlke

et al. 2018

Preprint

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Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing. Here, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions. * Measured at four times higher laser power (19.6 µW) than used in N&B measurements ** Average molecular brightness in N&B, interpolated to the same laser power (4.9 µW) for all red FPs

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“…Their reported DOL of 0.43±0.05 upon labeling under live-cell conditions differs substantially from previous reports where DOLs of 0.7-0.95 for SNAP-tag were determined using in vitro ensemble measurements with purified proteins [59,64]. Using a third approach where SNAP-tag was labeled after fixation and permeabilization of cells, Finan et al determined a DOL of around 0.7 for SNAPtag labeling with BG-Alexa647 [47]. To date, it is not known whether these differences in measured DOLs are caused by quantification approaches or represent actual differences in achieved DOLs due to sample preparation, cell lines, or the employed substrates.…”

Section: Labeling With Protein and Peptide Tagsmentioning

confidence: 55%

“…A fundamentally different approach is based on measuring the number of FPs for protein complexes with known oligomerization state. Such approaches were demonstrated to work with bacterial enzyme complexes that were expressed as FP fusions in mammalian cells [47], as well as for determining the maturation and photo-activation and photo-conversion efficiencies of photo-controllable FPs using plasma membrane receptors as templates [48].…”

Section: Labeling With Fluorescent Proteinsmentioning

confidence: 99%

Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting

Gruβmayer

Yserentant²,

Herten³

2019

Methods Appl. Fluoresc.

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The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in superresolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.

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A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Cited by 4 publications

References 20 publications

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting

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