Routing of Kv7.1 to endoplasmic reticulum plasma membrane junctions.

AIM: The voltage-gated Kv7.1 channel, in association with the regulatory subunit KCNE1, contributes to the IKs current in the heart. However, both proteins travel to the plasma membrane using different routes. While KCNE1 follows a classical Golgi-mediated anterograde pathway, Kv7.1 is located in endoplasmic reticulum-plasma membrane junctions (ER-PMjs), where it associates with KCNE1 before being delivered to the plasma membrane. METHODS: To characterize the channel routing to these spots we used a wide repertoire of methodologies, such as protein expression analysis (i.e. protein association and biotin labeling), confocal (i.e. immunocytochemistry, FRET, and FRAP), and dSTORM microscopy, transmission electron microscopy, proteomics, and electrophysiology. RESULTS: We demonstrated that Kv7.1 targeted ER-PMjs regardless of the origin or architecture of these structures. Kv2.1, a neuronal channel that also contributes to a cardiac action potential, and JPHs, involved in cardiac dyads, increased the number of ER-PMjs in nonexcitable cells, driving and increasing the level of Kv7.1 at the cell surface. Both ER-PMj inducers influenced channel function and dynamics, suggesting that different protein structures are formed. Although exhibiting no physical interaction, Kv7.1 resided in more condensed clusters (ring-shaped) with Kv2.1 than with JPH4. Moreover, we found that VAMPs and AMIGO, which are Kv2.1 ancillary proteins also associated with Kv7.1. Specially, VAP B, showed higher interaction with the channel when ER-PMjs were stimulated by Kv2.1. CONCLUSION: Our results indicated that Kv7.1 may bind to different structures of ER-PMjs that are induced by different mechanisms. This variable architecture can differentially affect the fate of cardiac Kv7.1 channels.

Cardiac multiscale bioimaging: from nano- through micro- to mesoscales.

Cardiac multiscale bioimaging is an emerging field that aims to provide a comprehensive understanding of the heart and its functions at various levels, from the molecular to the entire organ. It combines both physiologically and clinically relevant dimensions: from nano- and micrometer resolution imaging based on vibrational spectroscopy and high-resolution microscopy to assess molecular processes in cardiac cells and myocardial tissue, to mesoscale structural investigations to improve the understanding of cardiac (patho)physiology. Tailored super-resolution deep microscopy with advanced proteomic methods and hands-on experience are thus strategically combined to improve the quality of cardiovascular research and support future medical decision-making by gaining additional biomolecular information for translational and diagnostic applications.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes.

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

IP3R activity increases propensity of RyR-mediated sparks by elevating dyadic [Ca2＋].

Calcium (Ca2＋) plays a critical role in the excitation contraction coupling (ECC) process that mediates the contraction of cardiomyocytes during each heartbeat. While ryanodine receptors (RyRs) are the primary Ca2＋ channels responsible for generating the cell-wide Ca2＋ transients during ECC, Ca2＋ release, via inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are also reported in cardiomyocytes to elicit ECC-modulating effects. Recent studies suggest that the localization of IP3Rs at dyads grant their ability to modify the occurrence of Ca2＋ sparks (elementary Ca2＋ release events that constitute cell wide Ca2＋ releases associated with ECC) which may underlie their modulatory influence on ECC. Here, we aim to uncover the mechanism by which dyad-localized IP3Rs influence Ca2＋ spark dynamics. To this end, we developed a mathematical model of the dyad that incorporates the behaviour of IP3Rs, in addition to RyRs, to reveal the impact of their activity on local Ca2＋ handling and consequent Ca2＋ spark occurrence and its properties. Consistent with published experimental data, our model predicts that the propensity for Ca2＋ spark formation increases in the presence of IP3R activity. Our simulations support the hypothesis that IP3Rs elevate Ca2＋ in the dyad, sensitizing proximal RyRs towards activation and hence Ca2＋ spark formation. The stochasticity of IP3R gating is an important aspect of this mechanism. However, dyadic IP3R activity lowers the Ca2＋ available in the junctional sarcoplasmic reticulum (JSR) for release, thus resulting in Ca2＋ sparks with similar durations but lower amplitudes.

Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes.

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photo-activated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution Photo-Activated Localization Microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and "travelling" events that spread between neighbouring RyR clusters. Travelling sparks exhibited up to 8 distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute ß-adrenergic stimulation augments intra-cluster RyR recruitment to generate larger events. In contrast, RyR "dispersion" during heart failure facilitates the generation of travelling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Prolonged ß-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged ß-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of ß-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

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.

Functional Phenotype Flow Cytometry: On Chip Sorting of Individual Cells According to Responses to Stimuli.

The ability to effectively separate and isolate biological cells into specific and well-defined subpopulations is crucial for the advancement of our understanding of cellular heterogeneity and its relevance to living systems. Here is described the development of the functional phenotype flow cytometer (FPFC), a new device designed to separate cells on the basis of their in situ real-time phenotypic responses to stimuli. The FPFC performs a cascade of cell processing steps on a microfluidic platform: introduces biological cells one at a time into a solution of a biological reagent that acts as a stimulus, incubates the cells with the stimulus solution in a flow, and sorts the cells into subpopulations according to their phenotypic responses to the provided stimulus. The presented implementation of the FPFC uses intracellular fluorescence as a readout, incubates cells for 75 s, and operates at a throughput of up to 4 cells min-1 -resulting in the profiling and sorting of hundreds of cells within a few hours. The design and operation of the FPFC are validated by sorting cells from the human Burkitt's lymphoma cancerous cell line Ramos on the basis of their response to activation of the B cell antigen receptor (BCR) by a targeted monoclonal antibody.

Human Atrial Fibrillation Is Not Associated With Remodeling of Ryanodine Receptor Clusters.

The release of Ca2+ by ryanodine receptor (RyR2) channels is critical for cardiac function. However, abnormal RyR2 activity has been linked to the development of arrhythmias, including increased spontaneous Ca2+ release in human atrial fibrillation (AF). Clustering properties of RyR2 have been suggested to alter the activity of the channel, with remodeling of RyR2 clusters identified in pre-clinical models of AF and heart failure. Whether such remodeling occurs in human cardiac disease remains unclear. This study aimed to investigate the nanoscale organization of RyR2 clusters in AF patients - the first known study to examine this potential remodeling in diseased human cardiomyocytes. Right atrial appendage from cardiac surgery patients with paroxysmal or persistent AF, or without AF (non-AF) were examined using super-resolution (dSTORM) imaging. Significant atrial dilation and cardiomyocyte hypertrophy was observed in persistent AF patients compared to non-AF, with these two parameters significantly correlated. Interestingly, the clustering properties of RyR2 were remarkably unaltered in the AF patients. No significant differences were identified in cluster size (mean ∼18 RyR2 channels), density or channel packing within clusters between patient groups. The spatial organization of clusters throughout the cardiomyocyte was also unchanged across the groups. RyR2 clustering properties did not significantly correlate with patient characteristics. In this first study to examine nanoscale RyR2 organization in human cardiac disease, these findings indicate that RyR2 cluster remodeling is not an underlying mechanism contributing to altered channel function and subsequent arrhythmogenesis in human AF.

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy.

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA 'imagers' to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo.

Cell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

Ca2+ Release via IP3 Receptors Shapes the Cardiac Ca2+ Transient for Hypertrophic Signaling.

Calcium (Ca2+) plays a central role in mediating both contractile function and hypertrophic signaling in ventricular cardiomyocytes. L-type Ca2+ channels trigger release of Ca2+ from ryanodine receptors for cellular contraction, whereas signaling downstream of G-protein-coupled receptors stimulates Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs), engaging hypertrophic signaling pathways. Modulation of the amplitude, duration, and duty cycle of the cytosolic Ca2+ contraction signal and spatial localization have all been proposed to encode this hypertrophic signal. Given current knowledge of IP3Rs, we develop a model describing the effect of functional interaction (cross talk) between ryanodine receptor and IP3R channels on the Ca2+ transient and examine the sensitivity of the Ca2+ transient shape to properties of IP3R activation. A key result of our study is that IP3R activation increases Ca2+ transient duration for a broad range of IP3R properties, but the effect of IP3R activation on Ca2+ transient amplitude is dependent on IP3 concentration. Furthermore we demonstrate that IP3-mediated Ca2+ release in the cytosol increases the duty cycle of the Ca2+ transient, the fraction of the cycle for which [Ca2+] is elevated, across a broad range of parameter values and IP3 concentrations. When coupled to a model of downstream transcription factor (NFAT) activation, we demonstrate that there is a high correspondence between the Ca2+ transient duty cycle and the proportion of activated NFAT in the nucleus. These findings suggest increased cytosolic Ca2+ duty cycle as a plausible mechanism for IP3-dependent hypertrophic signaling via Ca2+-sensitive transcription factors such as NFAT in ventricular cardiomyocytes.

Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.

The unconventional biogenesis of Kv7.1-KCNE1 complexes.

The potassium channel Kv7.1 associates with the KCNE1 regulatory subunit to trigger cardiac I Ks currents. Although the Kv7.1/KCNE1 complex has received much attention, the subcellular compartment hosting the assembly is the subject of ongoing debate. Evidence suggests that the complex forms either earlier in the endoplasmic reticulum or directly at the plasma membrane. Kv7.1 and KCNE1 mutations, responsible for long QT syndromes, impair association and traffic, thereby altering I Ks currents. We found that Kv7.1 and KCNE1 do not assemble in the first stages of their biogenesis. Data support an unconventional secretory pathway for Kv7.1-KCNE1 that bypasses Golgi. This route targets channels to endoplasmic reticulum-plasma membrane junctions, where Kv7.1-KCNE1 assemble. This mechanism helps to resolve the ongoing controversy about the subcellular compartment hosting the association. Our results also provide new insights into I Ks channel localization at endoplasmic reticulum-plasma membrane junctions, highlighting an alternative anterograde trafficking mechanism for oligomeric ion channels.

In vitro reconstitution of branching microtubule nucleation.

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed 'branching' MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes.

Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches. We have used this technique successfully to purify both GFP-tagged and mCherry-tagged proteins, and their interacting partners, expressed in Drosophila melanogaster embryos. Although we demonstrate the efficacy of the GFP-binding protein and RFP-binding protein nanobodies from Chromotek, in theory any antibody could be coupled to the beads and used as a Cl-AP reagent.

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

Assessing Cardiomyocyte Excitation-Contraction Coupling Site Detection From Live Cell Imaging Using a Structurally-Realistic Computational Model of Calcium Release.

Calcium signaling plays a pivotal role in cardiomyocytes, coupling electrical excitation to mechanical contraction of the heart. Determining locations of active calcium release sites, and how their recruitment changes in response to stimuli and in disease states is therefore of central interest in cardiac physiology. Current algorithms for detecting release sites from live cell imaging data are however not easily validated against a known "ground truth," which makes interpretation of the output of such algorithms, in particular the degree of confidence in site detection, a challenging task. Computational models are capable of integrating findings from multiple sources into a consistent, predictive framework. In cellular physiology, such models have the potential to reveal structure and function beyond the temporal and spatial resolution limitations of individual experimental measurements. Here, we create a spatially detailed computational model of calcium release in an eight sarcomere section of a ventricular cardiomyocyte, using electron tomography reconstruction of cardiac ultrastructure and confocal imaging of protein localization. This provides a high-resolution model of calcium diffusion from intracellular stores, which can be used as a platform to simulate confocal fluorescence imaging in the context of known ground truth structures from the higher resolution model. We use this capability to evaluate the performance of a recently proposed method for detecting the functional response of calcium release sites in live cells. Model permutations reveal how calcium release site density and mitochondria acting as diffusion barriers impact the detection performance of the algorithm. We demonstrate that site density has the greatest impact on detection precision and recall, in particular affecting the effective detectable depth of sites in confocal data. Our findings provide guidance on how such detection algorithms may best be applied to experimental data and give insights into limitations when using two-dimensional microscopy images to analyse three-dimensional cellular structures.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.