scholarly journals Correlative super‐resolution light microscopy and electron microscopy determines spatial Ryanodine receptor type 2 distribution in mouse ventricular myocytes (LB707)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Tapaswini Das ◽  
Hiroyuki Hakozaki ◽  
Florentin Nitu ◽  
Donald Bers ◽  
Mark Ellisman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Super Resolution ◽  
Receptor Type

Junctophilin-2  physically interacts with ryanodine receptor type 2 for peripheral coupling of mouse cardiomyocytes

2020 ◽  
Author(s):  
Tianxia Luo ◽  
Ningning Yan ◽  
Mengru Xu ◽  
Fengjuan Dong ◽  
Qian Liang ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Bioinformatic Analysis ◽  
Cardiac Contraction ◽  
Receptor Type ◽  
Immunofluorescent Staining ◽  
Physical Interaction ◽  
C Terminus ◽  
Confocal Immunofluorescence ◽  
Intracellular Ca

Abstract Background: Ryanodine receptor type 2 (RyR2) mediate Ca 2+ release from the endoplasmic and sarcoplasmic reticulum (ER and SR), which is involved in the peripheral coupling of mouse cardiomyocytes, and thereby plays an important role in cardiac contraction. Junctophilin-2 (JPH2, JP2) is anchored to the plasma membrane (PM) and membranes of the ER and SR, and modulates intracellular Ca 2+ handling through regulation of RyR2. However, the potential RyR2 binding region of JPH2 is poorly understood. Methods: The interaction of JPH2 with RyR2 was studied using LC-MS/MS , bioinformatic analysis,co-immunoprecipitation studies in cardiac SR vesicles. GST-pull down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. Immunofluorescent staining was carried out to determine the colocalization of RyR2 and JPH2 in isolated mouse cardiomyocytes. Ion Optix photometry system was used to measure the levels of intracellular Ca 2+ transients in cardiomyocytes isolated from JPH2 knock down mice. Results: We report that (i) JPH2 interacts with RyR2 and (ii) the C terminus of the JPH2 protein can pull down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines. A decrease in the levels of JPH2 expression reduced the amplitude of Ca 2+ transients in cardiomyocytes. Conclusions: This study suggests that the C terminus domain of JPH2 is required for interactions with RyR2 in the context of peripheral coupling of mouse cardiomyocytes, which provide a molecular mechanism for looking for Ca 2+ - related diseases prevention strategies.

scholarly journals An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorothea Pinotsi ◽  
Simona Rodighiero ◽  
Silvia Campioni ◽  
Gabor Csucs
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Wide Field ◽  
Set Up ◽  
Bio Compatibility ◽  
Scanning Electron

Abstract A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling

Cell Calcium ◽  
2003 ◽  
Vol 34 (3) ◽  
pp. 261-269 ◽  
Author(s):  
Jens H Westhoff ◽  
Sung-Yong Hwang ◽  
R Scott Duncan ◽  
Fumiko Ozawa ◽  
Pompeo Volpe ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Intracellular Calcium ◽  
Ryanodine Receptor ◽  
Receptor Type ◽  
Intracellular Calcium Signaling ◽  
Homer Proteins

scholarly journals Ryanodine Receptor Type 2 Is Required for the Development of Pressure Overload-Induced Cardiac Hypertrophy

Hypertension ◽  
2011 ◽  
Vol 58 (6) ◽  
pp. 1099-1110 ◽  
Author(s):  
Yunzeng Zou ◽  
Yanyan Liang ◽  
Hui Gong ◽  
Ning Zhou ◽  
Hong Ma ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Ryanodine Receptor ◽  
Pressure Overload ◽  
Receptor Type

scholarly journals Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1

2017 ◽  
Vol 58 (6) ◽  
pp. 957-961 ◽  
Author(s):  
Zhiwen Ding ◽  
Jie Yuan ◽  
Yanyan Liang ◽  
Jian Wu ◽  
Hui Gong ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Cardiac Fibrosis ◽  
Mechanical Stretch ◽  
Receptor Type

scholarly journals Impaired local regulation of ryanodine receptor type 2 by protein phosphatase 1 promotes atrial fibrillation

2014 ◽  
Vol 103 (1) ◽  
pp. 178-187 ◽  
Author(s):  
David Y. Chiang ◽  
Na Li ◽  
Qiongling Wang ◽  
Katherina M. Alsina ◽  
Ann P. Quick ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ryanodine Receptor ◽  
Protein Phosphatase ◽  
Receptor Type ◽  
Protein Phosphatase 1 ◽  
Local Regulation

scholarly journals Impaired local regulation of ryanodine receptor type-2 by protein phosphatase 1 promotes atrial fibrillation

2013 ◽  
Vol 34 (suppl 1) ◽  
pp. P5016-P5016
Author(s):  
D. Y. Chiang ◽  
N. Li ◽  
G. Wang ◽  
Q. Wang ◽  
A. Quick ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ryanodine Receptor ◽  
Protein Phosphatase ◽  
Receptor Type ◽  
Protein Phosphatase 1 ◽  
Local Regulation

scholarly journals 3D visualisation of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

2021 ◽  
Author(s):  
Thomas M. D. Sheard ◽  
Miriam E. Hurley ◽  
Andrew J Smith ◽  
John Colyer ◽  
Ed White ◽  
...  
Keyword(s):  
Right Ventricular ◽  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Protein ◽  
3D Visualisation ◽  
Central Regions ◽  
Molecular Machinery ◽  
Receptor Clusters

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery in cardiomyocytes. Various adaptations of super-resolution microscopy have revealed intricate details of the structure, molecular composition and locations of these couplons. However, most optical super-resolution techniques lack the capacity for three-dimensional (3D) visualisation. Enhanced Expansion Microscopy (EExM) offers resolution (in-plane and axially) sufficient to spatially resolve individual proteins within peripheral couplons and within dyads located in the interior. We have combined immunocytochemistry and immunohistochemistry variations of EExM with 3D visualisation to examine the complex topologies, geometries and molecular sub-domains within RyR clusters. We observed that peripheral couplons exhibit variable co-clustering ratios and patterns between RyR and the structural protein, junctophilin-2 (JPH2). Dyads possessed sub-domains of JPH2 which occupied the central regions of the RyR cluster, whilst the poles were typically devoid of JPH2 and broader, and likely specialise in turnover and remodelling of the cluster. In right ventricular myocytes from rats with monocrotaline-induced right ventricular failure, we observed hallmarks of RyR cluster fragmentation accompanied by similar fragmentations of the JPH2 sub-domains. We hypothesise that the frayed morphology of RyRs in close proximity to fragmented JPH2 structural sub-domains may form the primordial foci of RyR mobilisation and dyad remodelling.

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