A5068412492- Advanced Fluorescence Microscopy Techniques
- Cardiac electrophysiology and arrhythmias
- Force Microscopy Techniques and Applications
- Advanced Electron Microscopy Techniques and Applications
- Cardiomyopathy and Myosin Studies
- Ion channel regulation and function
- Cell Image Analysis Techniques
- Neuroscience and Neural Engineering
- Advanced MRI Techniques and Applications
- Sex and Gender in Healthcare
- Cardiovascular Effects of Exercise
- Cellular Mechanics and Interactions
- Electron Spin Resonance Studies
- Nuclear Structure and Function
- Diversity and Career in Medicine
- Neuroscience and Neuropharmacology Research
- Nanofabrication and Lithography Techniques
- Cellular transport and secretion
- Advanced Biosensing Techniques and Applications
- 3D Printing in Biomedical Research
- Molecular Junctions and Nanostructures
- Caveolin-1 and cellular processes
- Muscle Physiology and Disorders
- Exercise and Physiological Responses
- Near-Field Optical Microscopy
University of Sheffield
2020-2024
UNSW Sydney
2023-2024
EMBL Australia
2023
Massey University
2023
University of Leeds
2016-2022
University of Exeter
2014-2018
The University of Queensland
2012-2014
University of Auckland
2009-2012
University of Aizu
2008
We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with mean size approximately 14 per cluster, which is almost order magnitude smaller than previously estimated. Clusters were typically not circular (as assumed) but elongated average aspect ratio 1.9. Edge-to-edge distances between adjacent RyR often <50...
Background Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It therefore develop practical methods that allow capturing full three-dimensional nature systems and also can visualize multiple species in same sample. Methodology/Principal Findings We show use a combination conventional near-infrared dyes, such as Alexa 647, 680 750, all excited with 671 nm diode laser,...
Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial visualizing these but limited by brightness markers, restricting the resolution and quantification individual within. Harnessing remarkable localization precision DNA-PAINT (<10 nm), we visualized punctate labeling...
Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as 'junctions', are fast communication hubs within excitable cells such neurones muscle. Here, we have examined two transgenic murine models probing role of junctophilin-2, a membrane-tethering protein crucial for formation molecular organisation sub-microscopic junctions in ventricular muscle heart. Quantitative single-molecule localisation microscopy showed that animals producing...
1. It is apparent from the literature that there are significant differences in excitation-contraction coupling between species, particularly density of calcium transporting proteins t-system and sarcoplasmic reticulum (SR) Ca(2+) release channels. Unfortunately, a lack information as to how principal structures link electrical excitation activation calcium-induced (CICR) different human animal models (particularly rat). 2. Comparison wheat germ agglutinin caveolin-3 labelling revealed...
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near cell surface. A majority nanodomains located deeper cells have remained unresolved due limited imaging depths and axial resolution modalities. series enhancements made expansion...
Quantitative understanding of the Ca(2+) handling in cardiac ventricular myocytes requires accurate knowledge ultrastructure and protein distribution. We have therefore developed high-resolution imaging analysis approaches to measure three-dimensional distribution immunolabelled proteins with confocal microscopy. Labelling single rat an antibody Z-line marker alpha-actinin revealed a complex architecture sarcomere misalignment across cells. Double immunolabelling was used relate structure...
Abstract High-force eccentric exercise results in sustained increases cytoplasmic Ca 2+ levels ([Ca ] cyto ), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters structure of membrane network inside fibres, tubular (t-) system, causing loss its predominantly transverse organization and an increase vacuolation longitudinal tubules across adjacent sarcomeres. The vacuoles displayed distinct -handling properties. Both t-system components...
Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, triads. Immunofluorescence staining fixed adult rat sections revealed clear differences between fast- and slow-twitch organization ryanodine...
Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes of proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory away from plasma membrane microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes subset P-selectin–negative WPBs....
Abstract Caveolae are small flask‐shaped invaginations of the surface membrane which proposed to recruit and co‐localize signaling molecules. The distinctive caveolar shape is achieved by oligomeric structural protein caveolin, three isoforms exist. Aside from finding that caveolin‐3 specifically expressed in muscle, functional differences between caveolin have not been rigorously investigated. Caveolin‐3 relatively cysteine‐rich compared caveolins 1 2, so we investigated its cysteine...
Skeletal muscle fibres are very large and elongated. In response to excitation there must be a rapid uniform release of Ca(2+) throughout for contraction. To ensure spread the fibre all sites, internalizes plasma membrane, form tubular (t-) system. Hence t-system forms complex dense network that is responsible excitation-contraction coupling other signalling mechanisms. However, we currently do not have detailed view this membrane because limitations in previously used imaging techniques...
Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery intracellular signalling in cardiomyocytes. While a range optical super-resolution microscopy techniques have revealed nanoscale structure these clusters, three-dimensional (3D) topologies clusters remained mostly unresolved. In this paper, we demonstrate exploitation molecular-scale resolution enhanced expansion (EExM) along with various 2D and 3D visualization strategies to observe topological...
Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect precision and accuracy. We present a simplified non-uniform noise model incorporates specific read-noise, offset sensitivity variation. Using this we develop new weighted least squared (WLS) fitting method designed remove the effect of non-uniformities. Simulations with model, performed test under which conditions corrections...