Photobleaching step analysis for robust determination of protein complex stoichiometries

Hummert, Johan; Yserentant, Klaus; Fink, Theresa; Euchner, Jonas; Herten, Dirk‐Peter

doi:10.1101/2020.08.26.268086

Cited by 11 publications

(34 citation statements)

References 51 publications

(58 reference statements)

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“…Photobleaching steps analysis (PBSA) was performed in Python using the quickPBSA package ( 32 ). The trace analysis algorithm in quickPBSA involves trace extraction for regions of interest (ROI), preliminary step detection for each trace, and iterative refinement using Bayesian marginal posterior ( 33 ).…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

Öz

Wang

Guérois

et al. 2021

Nucleic Acids Research

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We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

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Section: Methodsmentioning

confidence: 99%

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

Öz

Wang

Guérois

et al. 2021

Nucleic Acids Research

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“…Trace extraction and stepwise photobleaching analysis was performed using the python package quickpbsa. 18 In short, fluorescent spots were localised using the ImageJ 19 plugin 5…”

Section: Stepwise Photobleaching Analysismentioning

confidence: 99%

“…(Figure 3Aiii). 18 The number of discrete bleaching steps is equal to the number of eGFP molecules within the protein complex. Data representing all accepted spots for each receptor were pooled into photobleaching step frequency histograms ( Figure 3B).…”

Section: Stepwise Photobleaching Shows Gpvi Is Predominantly Monomerimentioning

confidence: 99%

“…The histograms display a range of photobleaching step frequencies in part because of the presence of non-fluorescent eGFP molecules within the protein complexes due to maturation issues or premature photobleaching, and the presence of overlapping fluorescent spots that cannot be resolved by the automated spot detection algorithms. 18 These occurrences could lead to underestimation or overestimation of bleaching steps and stoichiometry respectively. Therefore the distributions were modelled with functions taking into account a fraction of traces containing overlapping fluorescent spots (double spots) and non-fluorescent eGFP molecules.…”

Section: Stepwise Photobleaching Shows Gpvi Is Predominantly Monomerimentioning

confidence: 99%

“…If a protein is solely monomeric then stepwise photobleaching will result in complete loss of signal in a single spot, whereas it will result initially in partial loss for a dimeric protein. It is crucial to include controls of monomeric and dimeric proteins to enable calibration as results are influenced by overlapping signals and non-fluorescent tags 18. It is also critical to perform these studies on cells transfected with low levels of receptors to resolve individual monomers and dimers.…”

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confidence: 99%

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Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

et al. 2021

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Collagen has been proposed to bind to a unique epitope in dimeric GPVI and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerisation. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study we have used the advanced fluorescence microscopy techniques of single molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers, and that dimerisation through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and forms dimers in the membrane through diffusion giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

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Precision and Accuracy of Receptor Quantification on Synthetic and Biological Surfaces Using DNA-PAINT

et al. 2023

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Characterization of the number and distribution of biological molecules on 2D surfaces is of foremost importance in biology and biomedicine. Synthetic surfaces bearing recognition motifs are a cornerstone of biosensors, while receptors on the cell surface are critical/vital targets for the treatment of diseases. However, the techniques used to quantify their abundance are qualitative or semi-quantitative and usually lack sensitivity, accuracy, or precision. Detailed herein a simple and versatile workflow based on super-resolution microscopy (DNA-PAINT) was standardized to improve the quantification of the density and distribution of molecules on synthetic substrates and cell membranes. A detailed analysis of accuracy and precision of receptor quantification is presented, based on simulated and experimental data. We demonstrate enhanced accuracy and sensitivity by filtering out non-specific interactions and artifacts. While optimizing the workflow to provide faithful counting over a broad range of receptor densities. We validated the workflow by specifically quantifying the density of docking strands on a synthetic sensor surface and the densities of PD1 and EGF receptors (EGFR) on two cellular models.

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Photobleaching step analysis for robust determination of protein complex stoichiometries

Cited by 11 publications

References 51 publications

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

Precision and Accuracy of Receptor Quantification on Synthetic and Biological Surfaces Using DNA-PAINT

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