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.

scholarly journals Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

2015 ◽  
Vol 290 (33) ◽  
pp. 20477-20487 ◽  
Author(s):  
Florian Hiess ◽  
Alexander Vallmitjana ◽  
Ruiwu Wang ◽  
Hongqiang Cheng ◽  
Henk E. D. J. ter Keurs ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Receptor Clusters ◽  
And Function ◽  
Cardiac Ryanodine Receptor

scholarly journals Analysis of Cav1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

2010 ◽  
Vol 99 (12) ◽  
pp. 3923-3929 ◽  
Author(s):  
David R.L. Scriven ◽  
Parisa Asghari ◽  
Meredith N. Schulson ◽  
Edwin D.W. Moore
Keyword(s):  
Ryanodine Receptor ◽  
Ventricular Myocytes ◽  
Rat Ventricular Myocytes ◽  
Receptor Clusters

scholarly journals Super-resolution microscopy unveils FIP200-scaffolded, cup-shaped organization of mammalian autophagic initiation machinery

2019 ◽  
Author(s):  
Samuel J Kenny ◽  
Xuyan (Shirley) Chen ◽  
Liang Ge ◽  
Ke Xu
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Subcellular Location ◽  
Outer Face ◽  
Structural Protein ◽  
Er Exit Sites ◽  
Autophagy Pathway ◽  
20 Nm ◽  
Yeast Homolog ◽  
Super Resolution Microscopy

AbstractAutophagy is an essential physiological process by which eukaryotic cells degrade and recycle cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway have been identified, questions remain regarding the sequence, subcellular location, and structural requirements of autophagosome formation. Here, we characterize the structural organization of key components of the mammalian autophagic initiation machinery at ∼20 nm spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a structural asymmetry to enforce the directional recruitment of downstream components, including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we provide evidence that the early autophagic machinery is recruited in its entirety to these cup structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the phagophore membrane during elongation. We thus shed new light on the physical process of mammalian autophagic initiation and development at the nanometer-scale.

scholarly journals Three-Dimensional Distribution of Ryanodine Receptor Clusters in Cardiac Myocytes

2006 ◽  
Vol 91 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Ye Chen-Izu ◽  
Stacey L. McCulle ◽  
Chris W. Ward ◽  
Christian Soeller ◽  
Bryan M. Allen ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Three Dimensional ◽  
Dimensional Distribution ◽  
Receptor Clusters

scholarly journals Loss of dysferlin or myoferlin results in differential defects in excitation–contraction coupling in mouse skeletal muscle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Y. Barefield ◽  
Jordan J. Sell ◽  
Ibrahim Tahtah ◽  
Samuel D. Kearns ◽  
Elizabeth M. McNally ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Calcium Binding ◽  
Physiological Role ◽  
Muscle Disease ◽  
Calcium Handling ◽  
Membrane Repair ◽  
Tubule Formation ◽  
Ec Coupling ◽  
Associated Proteins ◽  
Receptor Clusters

AbstractMuscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.

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