In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

The stoichiometry of plasma membrane protein complexes is an important determinant of their function and of interactions between the individual proteins. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine protein stoichiometries in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine protein stoichiometries, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual signals are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high detection efficiency for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the stoichiometry of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require strong temporal separation of emission events within the same diffraction-limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the stoichiometries of two SLC26 multifunctional anion exchangers and to determine the stoichiometries of four members of the SLC17 family of organic anion transporters.

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Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy – high performance super-resolution at low cost

Scientific Reports ◽

10.1038/s41598-017-14762-6 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 20

Author(s):

Robin Diekmann ◽

Katharina Till ◽

Marcel Müller ◽

Matthias Simonis ◽

Mark Schüttpelz ◽

...

Keyword(s):

Single Molecule ◽

High Performance ◽

Low Cost ◽

Super Resolution ◽

Cmos Camera ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

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Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

10.1101/2021.12.01.469981 ◽

2021 ◽

Cited By ~ 1

Author(s):

David Virant ◽

Ilijana Vojnovic ◽

Jannik Winkelmeier ◽

Marc Endesfelder ◽

Bartosz Turkowyd ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

Single Molecule ◽

Spindle Pole Body ◽

Spindle Pole ◽

Centromeric Chromatin ◽

Localization Microscopy ◽

Process Understanding ◽

Segregation Process ◽

Single Molecule Localization Microscopy

AbstractThe key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far.In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

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Parameter-free molecular super-structures quantification in single-molecule localization microscopy

The Journal of Cell Biology ◽

10.1083/jcb.202010003 ◽

2021 ◽

Vol 220 (5) ◽

Author(s):

Mattia Marenda ◽

Elena Lazarova ◽

Sebastian van de Linde ◽

Nick Gilbert ◽

Davide Michieletto

Keyword(s):

Single Molecule ◽

Relevant Information ◽

Localization Microscopy ◽

Protein Clusters ◽

Formal Framework ◽

And Function ◽

Nuclear Ribonucleoprotein ◽

Molecular Components ◽

Single Molecule Localization Microscopy

Understanding biological function requires the identification and characterization of complex patterns of molecules. Single-molecule localization microscopy (SMLM) can quantitatively measure molecular components and interactions at resolutions far beyond the diffraction limit, but this information is only useful if these patterns can be quantified and interpreted. We provide a new approach for the analysis of SMLM data that develops the concept of structures and super-structures formed by interconnected elements, such as smaller protein clusters. Using a formal framework and a parameter-free algorithm, (super-)structures formed from smaller components are found to be abundant in classes of nuclear proteins, such as heterogeneous nuclear ribonucleoprotein particles (hnRNPs), but are absent from ceramides located in the plasma membrane. We suggest that mesoscopic structures formed by interconnected protein clusters are common within the nucleus and have an important role in the organization and function of the genome. Our algorithm, SuperStructure, can be used to analyze and explore complex SMLM data and extract functionally relevant information.

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Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽

10.1530/reprod/120.2.325 ◽

2000 ◽

pp. 325-335 ◽

Cited By ~ 29

Author(s):

A Calvo ◽

LM Pastor ◽

S Bonet ◽

E Pinart ◽

M Ventura

Keyword(s):

Ricinus Communis ◽

Lectin Histochemistry ◽

Apical Part ◽

Seminiferous Tubules ◽

In Situ Characterization ◽

Intense Staining ◽

Terminal Galactose ◽

Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

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Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

10.26434/chemrxiv.7992980.v1 ◽

2019 ◽

Author(s):

Zacharias Thiel ◽

Pablo Rivera-Fuentes

Keyword(s):

Subcellular Localization ◽

Fluorescent Probe ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Sensor ◽

Biological Functions ◽

Localization Microscopy ◽

Drug Activation ◽

Nitroreductase Activity ◽

Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

10.26434/chemrxiv.7992980 ◽

2019 ◽

Author(s):

Zacharias Thiel ◽

Pablo Rivera-Fuentes

Keyword(s):

Subcellular Localization ◽

Fluorescent Probe ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Sensor ◽

Biological Functions ◽

Localization Microscopy ◽

Drug Activation ◽

Nitroreductase Activity ◽

Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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