Fluorescence microscopy techniques, restricted by the diffraction limit of light, have seen a remarkable advancement in recent years. An approach called dSTORM (direct stochastic optical reconstruction microscopy) utilises the photoswitching capabilities of organic fluorophores when in the presence of special mounting media, the solution within which the sample is placed, to detect single molecule fluorescing events over time. The image that can be reconstructed from these events is not diffraction limited, but instead is limited by how well each event can be precisely localised. In Chapter 3 the importance of using a suitable mounting buffer in order to achieve super-resolution dSTORM is discussed in detail. A quantitative method for determining the reactivity of thiol dSTORM switching mountants was developed for use within the lab. Every fluorescent probe has different photophysical properties which can be manipulated by varying the composition of the switching buffer to enhance desirable qualities, such as; increased photon counts, faster switching rates, and longer survivability. In addition to investigating the effects of buffer composition the use of a near UV light-source was also explored as a means of manipulating the same properties to improve overall resolution and quality of the resulting images. A range of photoswitchable fluorescent dyes were tested including Alexa Fluor 660 which is a dye that to my knowledge has not been greatly tested for use in single molecule localisation microscopy by others to date. This dye performed strongly alongside the traditional Alexa Fluor 647 used for dSTORM imaging in optimal conditions. A relatively new approach to single molecule imaging which does not require the fluorophore to photoswitch, called DNA-PAINT (point accumulation for imaging in nanoscale topography), has been investigated throughout this thesis. This approach relies on the transient binding of small oligonucleotide sequences, called “Imagers”, to target docking strands anchored in positions of interest. These imagers have a photostable and bright fluorophore conjugated to the oligonucleotide. It is the transient immobilisation of the imager strand, as it binds to a fixed docking strand, which appears as stochastic blinks. The duration of these events, which can be extended by increasing the number of overlapping base pairs, is primarily responsible for improved localisation precision and therefore potentially overall resolution. At the end of Chapter 3 I compare this new pointillism microscopy approach, DNA-PAINT, with dSTORM using a set of custom-designed oligonucleotide sequences that allow both formats to be employed on the same target. The transient binding of small strands of oligonucleotides offers a far more controllable system for stochastic imaging. In Chapter 4 I use this superior approach to achieve greater resolution than other fluorescence techniques in biological samples, sufficient to visualise single ryanodine receptors (RyR). The RyR are extremely important in the contraction of muscle cells as they are capable of detecting transient changes to calcium concentration and are responsible for releasing large stores of calcium from the sarcoplasmic reticulum. With DNA-PAINT I observed that RyRs cluster into irregular arrays which contain significant gaps that are occupied by other proteins, including junctophilin (JPH). The stoichiometry of JPH with RyR varied cluster to cluster, exposing a new complexity in the regulation of RyRs. In Chapter 5, quantitative super-resolution is reliably achieved through the implementation of quantitative DNA-PAINT (qPAINT) within the Python Microscopy Environment (PYME) software. Quantitative measurements are possible because of the statistical predictability of DNA hybridisation and the near constant influx of fresh imager strands by diffusion. This results in limited photobleaching, a permanent dark state. The frequency with which a region of interest blinks is proportional to the number of binding sites available, and therefore the mean dark time between detected events is also inversely proportional. I validate my approach to qPAINT, which maintains the spatial information of individual structures, by using a DNA-origami test slide. Two distinguishable structures were present and an estimate for the ratio of available docking sites between them was satisfactorily established. I conclude that with this tool, molecule densities can be inferred and information about biological samples can be probed to new levels. The results of the full methodological approach to accomplish dual-colour super-resolution imaging of optically thick cardiac tissue, using both dSTORM and DNA PAINT techniques, is discussed in detail in Chapter 6. The current range of photoswitchable fluorophores limits the possible combination of molecular dyes for use with dSTORM and some compromise is made in their selection. For DNA-PAINT, the prospect of chromatic aberration is removed by imaging the same dye in subsequent rounds of imaging. The process, called Exchange-PAINT, allows the user to remove previously imaged imager strands, through a series of washes, and replace them with a complementary sequence for another target. I introduce the concept of using quencher strands to eliminate signal from unwanted imager sequences, accelerating their removal in samples of reduced diffusion and decreasing the risk of sample disturbance, in a process we termed Quencher Exchange-PAINT. Using this technique, I achieve superior super resolution results in optically thick samples. The results presented in this thesis are expected to (1) lead to a better understanding of the variables associated with single molecule localisation microscopy, (2) further reveal the complexity in cardiac protein distribution, (3) quantify relationships between co-localising proteins and other targets, and (4) apply DNA-PAINT to imaging in optically thick biological samples. This study shows promise for the future applications of the DNA-PAINT pointillism super-resolution method and its ability to investigate a multitude of biological questions.

Calcium induced calcium release (CICR) is the process mediating cardiac excitation contraction coupling (ECC). In brief, depolarisation of the plasma membrane of the cardiac myocyte leads to an influx of calcium (Ca2+) into the cytosol via the L-type voltage gated Ca2+ channels. The raised level of cytosolic Ca2+ initiates Ca2+ release from the junctional cisternae of sarcoplasmic reticulum (SR) through the opening of the ryanodine receptor 2 (RyR2). The exact mechanism of termination of CICR remains to be elucidated. It has been proposed that a drop in the luminal [Ca2+] reduces the open probability of RyR2 thereby leading to termination of CICR. It is also believed that RyR2 senses the luminal [Ca2+] through the formation of a quaternary complex with the SR proteins; calsequestrin (CSQ), triadin and junctin. However, the mechanism governing the assembly of this SR ‘luminal Ca2+ sensing complex’ is still far from being fully understood. A thorough knowledge of how this protein network is assembled is not only required for a robust understanding of the normal physiology of ECC but also for understanding the pathogenesis of disease, since disruption in luminal Ca2+ sensing is reported to lead to diastolic Ca2+ leak resulting in delayed after depolarisations (DADs), the precursor of premature beats and tachyarrhythmias. The primary focus of this thesis research was to investigate the structural basis for the formation of the luminal Ca2+ sensing complex with an emphasis on RyR, CSQ and triadin interactions. In order to achieve this goal, a protocol was developed to purify RyR2 from bovine heart employing a variety of techniques. Unfortunately this work resulted in only a partial purification of RyR2 with very low yields. However, more success was achieved with the isolation of the skeletal muscle ryanodine receptor isoform, RyR1, from sheep skeletal muscle employing sucrose gradient fractionation. The second aim of this study was to purify calsequestrin to enable investigations into its mode of interaction with the RyR. A molecular biology approach was taken and human cardiac calsequestrin (hCSQ2) was expressed as a GST tagged fusion protein and purified from E.coli BL21 (DE3) cells. A similar strategy was taken to express and purify the full-length and C-terminal luminal domain of mouse cardiac triadin isoform 1 (Trd1). However, this proved unsuccessful. A range of biochemical and biophysical techniques was next employed to examine whether the ryanodine receptor associated with hCSQ2 in the absence of triadin. It was found that purified RyR1 bound to immobilised GST-hCSQ2 through co-precipitation experiments suggesting a direct interaction between the two proteins. Further studies using surface plasmon resonance (SPR) also showed that immobilised hCSQ2 bound both RyR1 and RyR2. These findings were then developed by experiments employing quartz crystal microbalance and dissipation (QCM-D) monitoring. The data from QCM-D was also found to support a direct interaction of RyR1 (closed state) with both Ca2+ free and Ca2+ bound hCSQ2. Isolation of a RyR1 (closed state) and hCSQ2 complex (in the absence of Ca2+) was achieved using a sucrose cushion with an aliquot of the sample examined by transmission electron microscopy (TEM) using both negative staining and cryo-electron microscopy methods. The raw images of the complex suggested a direct interaction between RyR1 and CSQ2 in agreement with the data described above. However, intriguingly the hCSQ2 appeared to form strands of protein linking adjacent RyR molecules. These images, therefore, may suggest a possible role for hCSQ2 in a putative RyR coupled-gating mechanism. Another aspect of this research work was to optimise and employ a [3H] ryanodine binding assay to investigate how the channel activity of RyR1 and RyR2 within the SR preparations was regulated by hCSQ2 and triadin. Neither removal of endogenous CSQ from the SR membranes nor the addition of the recombinant hCSQ2, after removal of endogenous protein, modified the channel activity. However, interestingly, a synthesized domain of triadin (Trd KEKE motif) was found to enhance the channel activity as indicated by an increased [3H] ryanodine binding to both RyR1 and RyR2. In conclusion, the results from this thesis work provide evidence for a direct interaction between RyR and hCSQ2 and suggest a stimulatory role of a domain of triadin upon the activity of both isoforms of the ryanodine receptor.

Abnormal homeostasis of intracellular Ca2+ plays a deleterious role in muscle pathologies by triggering processes that lead to dysfunction and necrotic or apoptotic cell death. One pathology where there is significant Ca2+ induced cell damage is ischemia, which initiates further damage (also mediated by Ca2+) generated by the required treatment process of revascularization; namely ischemia-reperfusion injury. Pharmacological agents used therapeutically for cell protection, especially for cardiac protection in ischemic heart diseases, have only directly targeted one of the elements regulating Ca2+ homeostasis, the L-type Ca2+ channels (calcium channel blockers). Other agents, like beta blockers, indirectly target various elements, including sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and ryanodine receptors (RyRs). However, there are no pharmacological agents that directly and specifically target these two crucial elements required for intracellular SR Ca2+ homeostasis. Dr. Julio A. Copello’s group has previously studied the cardioprotective agent CGP-37157 (CGP), a benzothiazepine (BZT) derivative of the benzodiazepine (BZD) clonazepam. CGP was previously thought to decrease intracellular SR Ca2+ by acting as a blocker of the mitochondrial Na+/Ca2+ exchanger (Omelchenko et al., 2003). They found, however, that CGP also activates RyRs and inhibits the SERCA, which could better explain the SR effects of the drug (Neumann et al., 2011). These results suggest that drugs inducing partial depletion of SR Ca2+ stores could provide cellular protection in stressful circumstances or processes. The aims of the dissertation were organized based on the two processes that cause damage to muscle cells during ischemia: ischemia and subsequent reperfusion (ischemia-reperfusion injury) (Ibanez et al., 2015). Aim one and two focused on drug-protective action during the ischemic event, while aim three focused on drug protective action in the reperfusion (early post-ischemia) process. In the first Aim, experiments were designed to test the hypotheses that RyRs and/or SERCA could also be the target of i) Drugs with structural similarities to CGP (i.e., other BZTs and some BZDs) and ii) Drug known to confer cellular protection under stressful cellular conditions such as antiepileptic agents. We found that some BZTs (K201, CGP analog) and antiepileptic agents (Sipatrigine and Pimozide) demonstrated potential to prevent SR Ca2+ overload by inhibition of SERCA and, in some cases also by inducing mild activation of RyR channels. These results provided potential mechanisms of action for agents with cell protective action: targeting SERCA and preventing Ca2+ overload in pre-ischemia process. From the results of the first aim, K201 had the most significant effects in both SERCA inhibition and RyRs activation. Therefore, Aim 2 experiments focused on exploring with greater detail the action of the compound K201 on RyRs, SERCA and Ca2+ signaling. We found that K201 is a more potent SERCA blocker than RyR agonist and that SERCA inhibition remains under acidosis mimicking ischemic conditions. In Aim 3, the focus was on testing drugs with potential to prevent the overloaded SR from leaking Ca2+ (via RyRs) upon reperfusion. For that, we have examined various classes of organic polycationic agents in their ability to act as fast and reversible RyRs blockers. Currently, no agent with these characteristics is availableas a therapeutic or has been well defined for use as an experimental drug. The membrane permeable cation DHBP was identified as a potent RyR inhibitor with potential for rapid and transient inhibition of spontaneous SR Ca2+ release during reperfusion. In summary, we have defined the ability of some BZTs and antiepileptic agents (K201, CGP analog, Sipatrigine and Pimozide) to prevent/slow down SR Ca2+ overload by inhibition of SERCA, which may play an important role in their mechanisms of cell protection in ischemic events. In the case of BZT, these drugs may help their cause by producing mild activation of RyR2 channels, In addition, we have identify DHBP as a reversible and fast acting RyR inhibitor with potential as template for development of transient inhibitors of spontaneous SR Ca2+ release which may have significant protective action against injury during early reperfusion of the heart.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15408号 / 工博第3287号 / 新制||工||1495(附属図書館) / 27886 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Previous research showed mutations in muscle sarcoplasmic reticulum-bound calcium handler proteins cause swimming defects in embryonic zebrafish. CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed in all vertebrates has been defined to activate and inactivate multiple Ca2+ handler proteins involved in excitation- contraction coupling and relaxation of cardiac and skeletal muscle. In this study, evidence is provided through pharmacological and genetic intervention that CaMK-II inhibition and overexpression causes swimming defects, particularly response to stimuli and swimming ability, reinforced by immunolocalization of skeletal muscle. Transient CaMK-II inactivation does not have any long-term defects to swimming behavior. Overexpression of wild-type, constitutively active, and dominant-negative CaMK-II-GFP in embryos tended to co-localize in fast muscle which led to defects in swimming behavior. This study concludes that inhibition or overexpression of CaMK-II in skeletal muscle diminishes normal swimming behavior specifically in response to mechanical stimulation and swimming ability.

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.

Calcium is a ubiquitous second messenger in cells that plays a vital role in the control of cellular and physiological processes as diverse as cell division, memory and learning, fertilization and muscle contraction. Opening of the sarcoplasmic reticulum (SR) Ca2+-release channel, the ryanodine receptor (RyR), in response to mechanical or chemical stimuli via the dihydropyridine receptor (DHPR) is a crucial step in the process of muscle excitation-contraction coupling. I have determined the first high-resolution structure of a folded domain of RyR1 (RyR1A). The structure adopts a β-trefoil fold that is similar to the homologous suppressor domain of the inositol 1,4,5-trisphosphate receptor (IP3R). I identified a loop region in RyR1A concentrated with malignant hyperthermia (MH)- and central core disease (CCD)-associated mutations that have been implicated in perturbing inter-domain interactions with downstream regions of RyR. More recently I have used nuclear magnetic resonance (NMR) spectroscopy to study the structure and dynamics of the cardiac isoform (RyR2) A domain and its mutants. I detected a dynamic α-helix that undergoes an α-helix to β-strand switch in the catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated mutant, RyR2A Δ exon 3. This dynamic helix is localized at an interface with electron dense columns in the cryo-EM map of the tetrameric receptor that connect with the pore region, suggesting that this dynamic helix may also interact with downstream regions of RyR to gate the channel. My high-resolution structural studies in collaboration with others have shed light on the structural underpinnings of RyR function and dysfunction in human disease.

Calcium is a ubiquitous second messenger in cells that plays a vital role in the control of cellular and physiological processes as diverse as cell division, memory and learning, fertilization and muscle contraction. Opening of the sarcoplasmic reticulum (SR) Ca2+-release channel, the ryanodine receptor (RyR), in response to mechanical or chemical stimuli via the dihydropyridine receptor (DHPR) is a crucial step in the process of muscle excitation-contraction coupling. I have determined the first high-resolution structure of a folded domain of RyR1 (RyR1A). The structure adopts a β-trefoil fold that is similar to the homologous suppressor domain of the inositol 1,4,5-trisphosphate receptor (IP3R). I identified a loop region in RyR1A concentrated with malignant hyperthermia (MH)- and central core disease (CCD)-associated mutations that have been implicated in perturbing inter-domain interactions with downstream regions of RyR. More recently I have used nuclear magnetic resonance (NMR) spectroscopy to study the structure and dynamics of the cardiac isoform (RyR2) A domain and its mutants. I detected a dynamic α-helix that undergoes an α-helix to β-strand switch in the catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated mutant, RyR2A Δ exon 3. This dynamic helix is localized at an interface with electron dense columns in the cryo-EM map of the tetrameric receptor that connect with the pore region, suggesting that this dynamic helix may also interact with downstream regions of RyR to gate the channel. My high-resolution structural studies in collaboration with others have shed light on the structural underpinnings of RyR function and dysfunction in human disease.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from resource description page. Includes bibliographical references.