scholarly journals Cristae undergo continuous cycles of fusion and fission in a MICOS-dependent manner

2019 ◽  
Author(s):  
Arun Kumar Kondadi ◽  
Ruchika Anand ◽  
Sebastian Hänsch ◽  
Jennifer Urbach ◽  
Thomas Zobel ◽  
...  
Keyword(s):  
Imaging Techniques ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Stimulated Emission ◽  
Dependent Manner ◽  
Protein Markers ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Inner Membrane

AbstractThe mitochondrial inner membrane can reshape under different physiological conditions. How and at which frequency this occurs in vivo and what are the molecular players involved is unknown. Here we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that crista junctions (CJs) are dynamically fusing and dividing in a reversible and balanced manner at a timescale of seconds. CJ dynamics is strongly reduced in the absence of the MICOS subunit MIC13. Staining of the cristae membrane using different protein markers or two inner mitochondrial membrane-specific dyes revealed that cristae also undergo continuous cycles of fusion and fission. These processes are dependent on MIC13 and occur at a timescale of seconds, resembling CJ dynamics. Our data further suggest that MIC60 acts as a docking platform pioneering CJ formation. Overall, by employing a variety of advanced imaging techniques including FRAP (Fluorescence-Recovery-After Photobleaching), SPT (Single-Particle-Tracking), live-cell STED and confocal Airyscan microscopy we demonstrate that cristae undergo continuous cycles of fusion and fission in a manner that is mechanistically linked to CJ formation and dynamics.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals MINSTED fluorescence localization and nanoscopy

2021 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

Dynamics and Traffic for Transfecting Exogenous MicroRNA as Studied by Live-Cell Microscopy

2021 ◽  
Vol 17 (8) ◽  
pp. 1647-1653
Author(s):  
Ke Yang ◽  
Yuanyuan Wang ◽  
Bo Sun ◽  
Tian Tian ◽  
Zhu Dai ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Mean Square Displacement ◽  
Small Region ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Calculation Results ◽  
Long Time ◽  
Live Cell Microscopy ◽  
Cell Microscopy

MicroRNA (miRNA) has emerged as an important gene-regulator that shows great potential in gene therapy because of its unique roles in gene-regulation. However, the knowledge on their function and transportation in vivo is still lacking, and there are limited obvious evidences to define intracellular transportation of miRNA. In this study, the dynamics of exogenous miR-21 transfected into HeLa cells was traced by live-cell microscopy. Their transportation at key time points was recorded and dynamic properties were analyzed by single particle tracking (SPT) and mean square displacement (MSD) calculation. Results showed that the exogenous miRNAs bounded to cells quickly and went through lysosome into cytosol, where they were subsequently recruited into p-body. They finally were degraded, otherwise went back to cytosol in some way. Long time observation and analysis of motion mode showed that the miRNAs were confined in a small region and their motion modes were flexible in different intracellular microenvironment after entering the cells.

scholarly journals STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

2021 ◽  
Author(s):  
Pablo Carravilla ◽  
Anindita Dasgupta ◽  
Gaukhar Zhurgenbayeva ◽  
Dmytro I. Danylchuk ◽  
Andrey S. Klymchenko ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Spatial Resolution ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Live Cell ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Temporal And Spatial ◽  
Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

scholarly journals Title: In vivo phosphatidylserine variations steer Rho GTPase signaling in a cell-context dependent manner

2018 ◽  
Author(s):  
Matthieu Pierre Platre ◽  
Vincent Bayle ◽  
Laia Armengot ◽  
Joseph Bareille ◽  
Maria Mar Marques-Bueno ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Plant Root ◽  
Super Resolution ◽  
Small Gtpase ◽  
Auxin Signaling ◽  
Dependent Manner ◽  
Downstream Signaling

AbstractRho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

Photobleaching Reduction in Modulated Super Resolution Microscopy

Microscopy ◽  
2020 ◽  
Author(s):  
Jafar H Ghithan ◽  
Jennifer M Noel ◽  
Thomas J Roussel ◽  
Maureen A McCall ◽  
Bruce W Alphenaar ◽  
...  
Keyword(s):  
Continuous Wave ◽  
Imaging Techniques ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Optical Power ◽  
Stimulated Emission ◽  
Laser Beams ◽  
Synchronous Detection ◽  
Major Drawback ◽  
Sted Microscopy

Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases of photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive-index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

5224Endothelial autophagy triggers nuclear enrichment of miR-126-5p via a Mex3a-dependent pathway to confers endothelial protection and prevent atherosclerosis

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
D Santovito ◽  
L Natarelli ◽  
V Egea ◽  
K Bidzhekov ◽  
X Blanchet ◽  
...  
Keyword(s):  
Shear Stress ◽  
Protein Level ◽  
Rna Binding ◽  
Stimulated Emission ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
High Shear

Abstract Background MicroRNAs are versatile regulators of gene expression with profound implications for cardiovascular diseases and atherosclerosis. Among the most expressed in endothelial cells (ECs), the miR-126 duplex is crucial for angiogenesis and vascular protection, featuring different functional roles and homeostasis of the two strands. In line with separate activities, miR-126-3p and -5p undergo differential regulation in response to shear-stress in a KLF2-dependent manner. As both strands derive from a common precursor, the mechanism for strand specificity is unrelated to miRNA transcription and remains largely elusive as well as unknown is its relevance for vascular biology. Methods and results In human ECs (HUVECs) overexpression of KLF2 produced an up-regulation of the miR-126 precursor and miR-126-5p, but not miR-126-3p. Analysis of ECs overexpressing KLF2 or exposed to high-shear stress at transcriptional and protein level revealed the activation of the autophagic flux. Moreover, stimulation of autophagy by rapamycin replicated strand-specific regulation of miR-126. In particular, rapamycin promoted miR-126-3p degradation, while miR-126-5p was preserved and translocated to a nuclear reservoir complexed with the protein argonaute-2 (Ago2). Mutational scanning of fluorescently-labelled miR-126-5p revealed that nuclear shuttling required motifs distinct from the seed sequence. Size exclusion chromatography and Ago2-immunoprecipitation in autophagic ECs, as well as surface plasmon resonance (SPR) and electrophoretic mobility shift assays in vitro showed the formation of a ternary complex with the RNA-binding protein Mex3a. Preferential binding of miR-126-5p to Mex3a was confirmed by SPR, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Super-resolution microscopy by stimulated emission depletion (STED) visualized the Mex3a/Ago2 interaction on autophagosomal surfaces and silencing of Mex3a dampened rapamycin-induced nuclear miR-126-5p enrichment. Gain- and loss-of-function studies for miR-126-5p and Mex3a showed that Mex3a-guided nuclear miR-126-5p affect apoptosis mediators (e.g. caspase-3) at RNA and protein level, thus reducing the apoptotic rate. High-shear flow at non-predilection sites for atherosclerosis induced endothelial autophagy to promote nuclear enrichment of Ago2 and miR-126-5p in vivo in mice. Consistently, fewer nuclear Ago2 and miR-126-5p were observed in the endothelium of atheromatous area at bifurcations of human carotid arteries. These effects were abrogated in vivo by endothelial-specific deficiency in autophagy, thereby exacerbating atherosclerosis, and by Mex3a knock-out favouring apoptosis. Conclusion The autophagy-activated Mex3a-driven nuclear translocation represents a non-canonical mechanism by which miR-126-5p confer endothelial protection. Modulation of this pathway may open new opportunities for prevention and treatment of atherosclerosis.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Resolution Imaging ◽  
Membrane Protein Dynamics

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell–cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.

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