Nanoscale Organisation of Ryanodine Receptors and Junctophilin-2 in the Failing Human Heart

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 μm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

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Faculty Opinions recommendation of Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1052974.504926 ◽

2007 ◽

Author(s):

Mark Rose

Keyword(s):

Diffraction Limit ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction

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Faculty Opinions recommendation of Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1102184.558185 ◽

2008 ◽

Author(s):

Edward M Marcotte

Keyword(s):

Three Dimensional ◽

Super Resolution ◽

Stochastic Optical Reconstruction Microscopy ◽

Resolution Imaging ◽

Optical Reconstruction

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Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM)

Bioinformatics ◽

10.1093/bioinformatics/btab136 ◽

2021 ◽

Author(s):

Lekha Patel ◽

David Williamson ◽

Dylan M Owen ◽

Edward A K Cohen

Keyword(s):

Probability Distribution ◽

Single Molecule ◽

Immunological Synapse ◽

Training Data ◽

Supplementary Information ◽

Cellular Structures ◽

Exact Probability ◽

Stochastic Optical Reconstruction Microscopy ◽

Exact Probability Distribution ◽

Optical Reconstruction

Abstract Motivation Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Results Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Availability and implementation Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary information Supplementary data are available at Bioinformatics online.

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Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902440116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18423-18428 ◽

Cited By ~ 20

Author(s):

Huizhong Xu ◽

Zhisong Tong ◽

Qing Ye ◽

Tengqian Sun ◽

Zhenmin Hong ◽

...

Keyword(s):

Meiotic Chromosome ◽

Molecular Organization ◽

Homologous Chromosomes ◽

Meiotic Chromosomes ◽

C Terminus ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction ◽

20 Nm ◽

Chromosome Axis

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure’s lateral elements (LEs). While the components of the mammalian chromosome axis/LE—including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2—are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

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Mapping Neuronal Connectivity Using Stochastic Optical Reconstruction Microscopy (Storm): The Brainstorm Project

Biophysical Journal ◽

10.1016/j.bpj.2009.12.1153 ◽

2010 ◽

Vol 98 (3) ◽

pp. 214a

Author(s):

Melike Lakadamyali ◽

Mark Bates ◽

Hazen Babcock ◽

Jeff Lichtman ◽

Xiaowei Zhuang

Keyword(s):

Neuronal Connectivity ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction

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Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ε to RACK2 in adult rat and mouse

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00247.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. H718-H725 ◽

Cited By ~ 6

Author(s):

Richard A. Fenton ◽

Satoshi Komatsu ◽

Mitsuo Ikebe ◽

Lynne G. Shea ◽

James G. Dobson

Keyword(s):

Confocal Microscopy ◽

Phospholipase C ◽

Adrenergic Agonist ◽

Rat Cardiomyocytes ◽

Scanning Confocal Microscopy ◽

Adult Rat ◽

Sds Page ◽

T Tubules ◽

Isolated Rat ◽

Stimulation Of

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ε to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ε induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ε and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ε to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ε co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ε with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ε translocation and association with RACK2.

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Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

Scientific Reports ◽

10.1038/s41598-018-35297-4 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 2

Author(s):

Ruiheng Li ◽

Pantelis Georgiades ◽

Henry Cox ◽

Sorasak Phanphak ◽

Ian S. Roberts ◽

...

Keyword(s):

Graphene Oxide ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction

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Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI)

Neuropathology and Applied Neurobiology ◽

10.1111/nan.12426 ◽

2017 ◽

Vol 44 (4) ◽

pp. 417-426 ◽

Cited By ~ 6

Author(s):

A. H. Hainsworth ◽

S. Lee ◽

P. Foot ◽

A. Patel ◽

W. W. Poon ◽

...

Keyword(s):

White Matter ◽

Super Resolution ◽

Subcortical White Matter ◽

Stochastic Optical Reconstruction Microscopy ◽

Resolution Imaging ◽

Optical Reconstruction

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Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.4.c1221 ◽

1996 ◽

Vol 270 (4) ◽

pp. C1221-C1227 ◽

Cited By ~ 74

Author(s):

A. A. McDonough ◽

Y. Zhang ◽

V. Shin ◽

J. S. Frank

Keyword(s):

Guinea Pig ◽

Driving Force ◽

Human Heart ◽

Sodium Pump ◽

Immunogold Labeling ◽

Guinea Pig Heart ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Na Pump ◽

T Tubules

The cardiac Na+ pump (Na+ -K+ -ATPase) provides the driving force for the Na+/Ca2+ exchanger, a determinant of intracellular Ca2+ stores. Three Na+ pump alpha-catalytic subunit isoforms are expressed in human heart, alpha1 and alpha2 are expressed in rat heart, and only alpha1 is expressed in guinea pig heart. The objective of this study was to determine whether there are isoform-specific patterns of expression in the transverse tubules (T tubules) vs. the peripheral sarcolemma. In adult rat cardiomyocytes, anti-alpha1-specific antibodies labeled the T tubules more intensely than the peripheral sarcolemma, in which labeling was patchy, the same pattern reported for distribution of the Na+/Ca2+ exchanger (J. S. Frank, G. Mottino, D. Reid, R. S. Molday, and K. D. Philipson, J. Cell Biol. 117: 337-345, 1992), whereas anti-alpha2- and anti-beta1-antibodies uniformly labeled T tubules and peripheral sarcolemma. In guinea pig cardiomyocytes, an anti-alpha-antibody against an extracellular epitope evenly labeled the peripheral sarcolemma and T tubules, and immunogold labeling demonstrated coincidence of alpha-subunits and intramembranous particles in sarcolemma. In summary, Na+ pumps are located in both peripheral sarcolemma and T tubules of cardiomyocytes expressing either multiple or single Na+ pump isoforms.

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