scholarly journals Dissecting the intracellular signalling and fate of a DNA nanosensor by super-resolution and quantitative microscopy

Nanoscale ◽  
2020 ◽  
Vol 12 (28) ◽  
pp. 15402-15413
Author(s):  
Agata Glab ◽  
Alessandro Bertucci ◽  
Fabiana Martino ◽  
Marcin Wojnilowicz ◽  
Alessia Amodio ◽  
...  
Keyword(s):  
Super Resolution ◽  
Intracellular Signalling ◽  
Quantitative Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

The intracellular interactions and fate of a DNA nanosensor were investigated by combining quantitative microscopy and stochastic optical reconstruction microscopy.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

2020 ◽  
Vol 12 (535) ◽  
pp. eaay0071 ◽  
Author(s):  
Zhen Liu ◽  
Quynh P. H. Nguyen ◽  
Qingxu Guan ◽  
Alexandra Albulescu ◽  
Lauren Erdman ◽  
...  
Keyword(s):  
Lung Function ◽  
A Priori ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Defects ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Motile Cilia ◽  
Optical Reconstruction

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

Nanoscale ◽  
2015 ◽  
Vol 7 (6) ◽  
pp. 2511-2519 ◽  
Author(s):  
Jing Gao ◽  
Ye Wang ◽  
Mingjun Cai ◽  
Yangang Pan ◽  
Haijiao Xu ◽  
...  
Keyword(s):  
Distribution Pattern ◽  
Lipid Rafts ◽  
Essential Role ◽  
Super Resolution ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Polarized Cells ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Cluster Maintenance

We investigate the distribution of membrane EGFR by direct stochastic optical reconstruction microscopy (dSTORM). Our results illustrate the clustering distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.

scholarly journals Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

2020 ◽  
Author(s):  
Paramita Ray ◽  
Krishnan Raghunathan ◽  
Aarif Ahsan ◽  
Uday Sankar Allam ◽  
Shirish Shukla ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Super Resolution ◽  
Receptor Internalization ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Steady State Levels ◽  
Epidermal Growth ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

ABSTRACTWe previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

scholarly journals Volumetric super-resolution imaging by serial ultrasectioning and stochastic optical reconstruction microscopy in mouse neural tissue

2021 ◽  
Vol 2 (4) ◽  
pp. 100971
Author(s):  
Tarlan Vatan ◽  
Jacqueline A. Minehart ◽  
Chenghang Zhang ◽  
Vatsal Agarwal ◽  
Jerry Yang ◽  
...  
Keyword(s):  
Super Resolution ◽  
Neural Tissue ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction

scholarly journals Surface Charge Manipulation and Electrostatic Immobilization of Synaptosomes for Super-resolution Imaging: A Study on Tau Compartmentalization

2021 ◽  
Author(s):  
Ushashi Bhattacharya ◽  
Jia-Fong Jhou ◽  
Yi-Fong Zou ◽  
Gerald Abrigo ◽  
Shu-Wei Lin ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Protein Localization ◽  
Super Resolution ◽  
Synaptic Terminals ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Neural Transmission ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Cortical Synapses ◽  
Induced Aggregation

Abstract Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a simple and facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

scholarly journals Vectashield-induced fluorescence quenching hinders conventional and super resolution microscopy

2018 ◽  
Author(s):  
Aleksandra Arsić ◽  
Nevena Stajković ◽  
Rainer Spiegel ◽  
Ivana Nikić-Spiegel
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Practical Advice ◽  
Alexa Fluor ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Conventional Microscopy ◽  
Optical Reconstruction ◽  
The Right ◽  
Super Resolution Microscopy

AbstractFinding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent ON state. This helps identifying structures of interests. Subsequently, labelled samples are brought to blinking, which is necessary for localization of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor (AF) 647, blinks in Vectashield. However, after adding Vectashield to our samples, we noticed that the fluorescence intensity of AF647 and its improved variant, AF647+, is quenched. Since structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) practical advice on how to use Vectashield for dSTORM imaging of AF647 and AF647+.

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