Automated Analysis of Single-Molecule Photobleaching Data by Statistical Modeling of Spot Populations

Liesche, Clarissa; Grußmayer, Kristin S.; Ludwig, Michael; Wörz, Stefan; Rohr, Karl; Herten, Dirk‐Peter; Beaudouin, Joël; Eils, Roland

doi:10.1016/j.bpj.2015.10.035

Cited by 33 publications

(40 citation statements)

References 38 publications

(54 reference statements)

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“…The slower the decrease of the number of spots relative to the decrease of the intensity, the larger the number of fluorophores per spot. 262 Yet another approach is to count molecules by means of photon arrival statistics. 263,264 This technique exploits the photon antibunching effect, which essentially states that a single emitting quantum system (a fluorophore in this case) emits photons one at a time.…”

Section: The Counting Problemmentioning

confidence: 99%

Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis

Lee

Tsekouras

Calderon³

et al. 2017

Chem. Rev.

141

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Super-resolution microscopy provides direct insight into fundamental biological processes occurring at length scales smaller than light’s diffraction limit. The analysis of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data analysis methods starting from an overview of basic statistical techniques underlying the analysis of super-resolution and, more broadly, imaging data. We subsequently break down the analysis of super-resolution data into four problems: the localization problem, the counting problem, the linking problem, and what we’ve termed the interpretation problem.

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Section: The Counting Problemmentioning

confidence: 99%

Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis

Lee

Tsekouras

Calderon³

et al. 2017

Chem. Rev.

141

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“…including prior information about dye photo-physics [136]. Here, published datasets were analyzed, [137]. Counting the number of photo-bleaching steps has been extensively used to quantify membrane receptors and ion channels in the plasma membrane with typical stoichiometries on the order of 4-5 [11].…”

Section: Intensity-based Counting (Ibc)mentioning

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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“…To get insights into this process, we compared two different recombinant ligands, the natural soluble CD95L (sCD95L), a weak apoptosis inducer (23), and the isoleucine zipper-fused ligand (IZ-sCD95L), which is a strong apoptosis inducer (29) (53). We have previously shown that sCD95L and IZ-sCD95L are trimeric at the population level even in the picomolar range (27), a result that we validated here by comparing the brightness of mCherry-sCD95L, mCherry-IZ-sCD95L and mCherry-IZ to the one of mCherry alone by fluorescence correlation spectroscopy (FCS) (Fig. S7A).…”

Section: Equal Cd95 Receptor Occupancy With Scd95l and Iz-scd95l Tranmentioning

confidence: 99%

“…The extracellular domain of CD95L can be cleaved by proteases (20) (21) (22) and the soluble product, sCD95L, is a weak inducer of apoptosis that can block the activity of the membrane form (23) (20) (24) (25). Using gel filtration (25), X-ray crystallography (26) and single molecule fluorescence microscopy (27) it was shown that sCD95L forms a trimer. Two types of modifications can transform sCD95L into a strong apoptosis inducer: its secondary multimerization using antibodies (24) (28) or its recombinant fusion to a trimerization domain, like the isoleucine zipper (IZ) (29) or the foldon that is part of the protein fibritin from the bacteriophage T4 (30).…”

Section: Introductionmentioning

confidence: 99%

CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

Liesche

Berndt

Fricke

et al. 2018

Preprint

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Keywords: quantitative fluorescence microscopy, FRET, FRAP, PALM, single transmembrane receptor activation, receptor oligomerization, CD95/Fas, apoptosis Highlights:• At a density of less than 10 receptors per µm 2 CD95 exists as monomer (58%) and dimer (42%)• Pre-formed dimers do not contribute to ligand-induced CD95 apoptotic signaling • The PLAD of CD95 attenuates overexpression-induced, ligand-independent cell death • soluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitment • FADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity Abstract CD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Förster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using timeresolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.

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Automated Analysis of Single-Molecule Photobleaching Data by Statistical Modeling of Spot Populations

Cited by 33 publications

References 38 publications

Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis

Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis

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

CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

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