Single molecule localization deep within thick cells; a novel super‐resolution microscope

Tafteh, Reza; Scriven, David R.L.; Moore, Edwin D.W.; Chou, Kuang-Chao

doi:10.1002/jbio.201500140

Cited by 19 publications

(26 citation statements)

References 24 publications

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“… a ; Tafteh et al . ), our calibration method showed consistent estimation of RyR cluster and CRU sizes at internal sites up to a depth of ∼6 μm (Fig. H and I ).…”

Section: Discussionsupporting

confidence: 63%

“…One major advantage of our current 3D event-based approach is that it allows for the quantification of interior clusters deeper beneath the cell surface. Although the J Physiol 597.2 accuracy of z-localization is depth dependent (Baddeley et al 2011a;Tafteh et al 2016), our calibration method showed consistent estimation of RyR cluster and CRU sizes at internal sites up to a depth of ß6 μm (Fig. 5H and I).…”

Section: D Surface Vs Interior Clusters: Functional Implicationsmentioning

confidence: 72%

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3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Shen

Brink

Hou

et al. 2018

The Journal of Physiology

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Key points Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non‐dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z‐axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. Abstract Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

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“… a ; Tafteh et al . ), our calibration method showed consistent estimation of RyR cluster and CRU sizes at internal sites up to a depth of ∼6 μm (Fig. H and I ).…”

Section: Discussionsupporting

confidence: 63%

Section: D Surface Vs Interior Clusters: Functional Implicationsmentioning

confidence: 72%

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Shen

Brink

Hou

et al. 2018

The Journal of Physiology

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“…Phosphorylation increases cardiac contractility by, in part, increasing the likelihood that the RyR2 channel will open, but how this is accomplished is poorly understood. We used dSTORM to image RyR2 proteins [11], from which we constructed a 3D point-cloud. To limit the computational times we first segmented the images to isolate Z-lines (9 from the control cells and 14 from those that were phosphorylated; between 30,000 and 65,000 blinks each), which is where jSR and RyR2 are primarily located.…”

Section: Methodsmentioning

confidence: 99%

Sub:Cellular Network Analysis of Ryanodine Receptor Positioning in Control and Phosphorylated States

Khater

Scriven

Moore

et al. 2016

2016 Computing in Cardiology Conference (CinC)

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Type 2 ryanodine receptors (RyR2) are large (mass=2.2 MegaDalton) proteins expressed in heart cells that mediate the release of calcium ions (Ca 2+ ) that, in turn, modulate contraction of the heart. In this work, we analyze the sub-cellular spatial distribution of RyR2 using data from superresolution microscopy, an imaging technique that allows highly accurate positioning (<10 nm in x and y; <40 nm in z) of the RyR2 within the heart muscle cell. In particular, we present the first work to examine network measures of extracted RyR2 locations to examine the clustering behaviour of these channels. We collected images from two groups of healthy cardiac cells; the control consisted of 8 cells, while the second group of 8 cells were treated with a chemical cocktail to phosphorylate the RyR2 and to inhibit dephosphorylation. We examined the classification accuracy (using random forest classifier) and the group differences (using Mann-Whitney statistical test and Bonferroni multiple comparison correction) based on several network measures, at multiple proximity thresholds. Several network measures we examined revealed features (e.g. network clustering) that enabled us to differentiate between these two populations (p<0.00045) with high classification accuracy (>95% at proximity thresholds 200 and 250 nm). Our findings may help in better understanding Ca 2+ signaling during contraction and give insight into the changes that underlie its regulation. IntroductionThe process of calcium release is essential for subcellular dynamic functions such as excitation-contraction (E-C) coupling in cardiac myocytes. The ryanodine receptors (RyRs) are a class of large protein channels responsible for mediating the rapid release of calcium ions (Ca 2+ ) from intracellular stores into the cytosol [1]. There are multiple isoforms of RyR proteins: RyR1 is primarily expressed in skeletal tissues. RyR2 is primarily expressed in cardiac myocytes and RyR3 is widely expressed in the brain. In this paper, we focus on cardiac cells and on RyR2 proteins in the dyad. The dyad is the structural element formed by the close apposition of the membranes of the sarcolemma (either surface or t-tubule) and the junctional sarcoplasmic reticulum (jSR) [2]. RyR2 proteins are distributed on the jSR membrane opposite the L-type channels in the sarcolemma.The principal determinant of the force of cardiac contraction is the amount of Ca 2+ released from the sarcoplasmic reticulum, and this is controlled in part by how RyR2 are distributed relative to each other. Electron tomography has demonstrated that the RyR2 distribution can be altered by physiological factors, such as phosphorylation (the fight or flight response) [3].The recent development of superresolution microscopy (also called nanoscopy), such as direct stochastic optical reconstruction microscopy dSTORM, can bring new insight into cardiac cell biology. At a high level, dSTORM nanoscopy offers single molecule localization and allows for nanometer-precision identification of RyR2 locations, w...

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“…To enhance localization precision, we used an SMLM microscope equipped with real-time nanometer-scale drift correction hardware 29 and included only high-precision localizations to improve localization accuracy 30,31 . With this inhouse built SMLM microscope, the localization precision approaches 10 nm 32 , and the drift is limited to 1 nm in the x-y plane and 3 nm in the z axis 29,33 . Modularity analysis and group matching show that S1A scaffolds can dimerize to form S1B scaffolds and oligomerize to form hemispherical S2 scaffolds.…”

Section: Introductionmentioning

confidence: 99%

Super-resolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae

Khater

Liu

Chou

et al. 2018

Preprint

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Caveolin-1 (Cav1), the coat protein for caveolae, also forms non-caveolar Cav1 scaffolds. Single molecule Cav1 super-resolution microscopy analysis previously identified caveolae and three distinct scaffold domains: smaller S1A and S2B scaffolds and larger hemispherical S2 scaffolds. Application here of network modularity analysis of SMLM data for endogenous Cav1 labeling in HeLa cells shows that small scaffolds combine to form larger scaffolds and caveolae. We find modules within Cav1 blobs by maximizing the intra-connectivity between Cav1 molecules within a module and minimizing the inter-connectivity between Cav1 molecules across modules, which is achieved via spectral decomposition of the localizations adjacency matrix. Features of modules are then matched with intact blobs to find the similarity between the module-blob pairs of group centers. Our results show that smaller S1A and S1B scaffolds are made up of small polygons, that S1B scaffolds correspond to S1A scaffold dimers and that caveolae and hemi-spherical S2 scaffolds are complex, modular structures formed from S1B and S1A scaffolds, respectively. Polyhedral interactions of Cav1 oligomers therefore leads progressively to the formation of larger and more complex scaffold domains and the biogenesis of caveolae.

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Single molecule localization deep within thick cells; a novel super‐resolution microscope

Cited by 19 publications

References 24 publications

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Sub:Cellular Network Analysis of Ryanodine Receptor Positioning in Control and Phosphorylated States

Super-resolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae

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