Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

Wong, Joseph; Baddeley, David; Bushong, Eric A.; Yu, Zeyun; Ellisman, Mark H.; Hoshijima, Masahiko; Soeller, Christian

doi:10.1016/j.bpj.2013.02.059

Cited by 65 publications

(50 citation statements)

References 11 publications

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“…Extended RyR patches surrounded by areas occupied by myofibrils (which was confirmed in double labelling images with α-actinin, Fig. S1A-C), similar to previous observations in both rat and mouse myocytes Wong et al, 2013). Closer inspection revealed differences between cells from animal models in which JPH2 expression was altered.…”

Section: Ryr Clusters In Myocytes With Altered Jph2 Expressionsupporting

confidence: 90%

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

Munro

Jayasinghe

Wang

et al. 2016

Journal of Cell Science

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Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as ‘junctions’, are fast communication hubs within excitable cells such as neurones and muscle. Here we have examined two transgenic murine models probing the role of junctophilin-2, a membrane tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR). Although this change was associated with much enlarged RyR clusters that due to their size should be more excitable, functionally it caused a mild inhibition in the calcium signalling output of the junctions (calcium sparks). Analysis of the single molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the calcium signalling properties of the junction and thus the contractile function of the cell.

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Section: Ryr Clusters In Myocytes With Altered Jph2 Expressionsupporting

confidence: 90%

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

Munro

Jayasinghe

Wang

et al. 2016

Journal of Cell Science

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“…49 These data volumes demonstrate the relative differences in the tubule widths between sheep and rat t-tubules and, for the first time, resolve local variations in tubule diameters that were previously not directly visualised in fluorescence micrographs due to the diffraction limit (although see our previous discussion of rat t-tubules, 39 Sache et al 41 of rabbit; see arrowheads in Fig 3C-ii for example dSTORM image). Similar nanometre-scale dilatations were observed in the junctional segments of mouse ventricular t-tubules using tomographic EM and dSTORM data 50 (arrowheads in Fig 3A-ii). More recent tomographic reconstructions suggest that these may be folds of the t-tubules that putatively improves the contact between RyR and the trigger.…”

Section: Progress In Sub-micron-scale Microscopy Of Cardiac and Skelesupporting

confidence: 51%

“…Correlative or supplemented imaging SMLM samples with EM, whenever possible, promises additional information such as 3D nanostructure and relationship with other organelles as well as a means of validating the nano-scale measurements, e.g. 50 We also underscore the potential utility of SMLM and other optical super-resolution modes in supplementing (diffraction-limited) confocal fluorescence microscopy of calcium micro-and nanodomains (e.g. imaging Ca 2+ sparks with intra-dyadic Ca 2+ sensors 87 ) to correlate functional measurements with the local ultrastructure.…”

Section: A Case For Improving Imaging Methodsmentioning

confidence: 99%

Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

et al. 2014

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The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the ttubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

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“…Regions of t-t dilation have recently been identified as a feature of mice t-ts. 27 Figure 1C shows an enlarged portion of the myocyte, illustrating the periodicity of t-ts as they extend out from the sarcolemma. The t-ts do not generally form contacts or fuse with an adjacent invagination, as illustrated in inset to Figure 1C.…”

Section: Sheep T-t Morphologymentioning

confidence: 99%

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules

Pinali

Bennett

Davenport

et al. 2013

Circulation Research

124

127

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T ransverse-tubules (t-ts) are invaginations of the plasma membrane, which in cardiac muscle facilitate transmission of the action potential from the exterior to interior of the cell. Dyads or couplons are formed along regions of the t-ts by the close apposition of the junctional portion of the sarcoplasmic reticulum (jSR). It is within the compartment formed by these 2 structures, the dyadic cleft, that the contractile function in the heart is regulated by the interplay between the L-type voltage-gated Ca 2+ channel (LTCC) localized to the t-ts and the ryanodine receptor (RyR2) anchored in the jSR.1 Critical to this regulation is the arrangement of the jSR and t-t membranes, which are held in a precise geometric organization 2 separated by a gap of 12 to 15 nm. Changes to the microanatomy between the 2 membranes, and hence the spatial relationship between the LTCCs and RyR2s, are associated with Ca 2+ handling abnormalities and impaired contractile function, 3 although the effect on systolic Ca 2+ remains equivocal. 4 Our current understanding of the 3-dimensional (3D) organization of t-ts is mainly derived from studies using confocal microscopy. [5][6][7] Elegant experiments have revealed that the t-ts form regular arrays with a periodicity that correlates to the position of the Z-lines. 8 The dimensions and branching patterns are varied and species-dependent. For example, rat myocytes are shown to have a more geometrically complex arrangement compared with the human t-t system, which has a radial distribution within the cell, akin to the spokes of a wheel. 9 Recently, the development of stimulated emission depletion microscopy has pushed the resolution frontier to ≈60 nm in the focal plane, providing ultrastructural details of the t-t system in control and failing rat hearts showing Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation-contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure.Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results:Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collap...

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Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

Cited by 65 publications

References 11 publications

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules

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