New review chapter: “Advances in the Visualization of Molecular Assemblies Within Cellular Signaling Nanodomains: Insights From a Decade of Mapping of Ryanodine Receptor Clusters”

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Nanodomains are naturally assembled signaling stations, which facilitate fast and highly regulated signaling within and between cells. Calcium (Ca2+) nanodomains known as junctional membrane complexes (JMCs) transduce fast and highly synchronized intracellular signals, which are required by a variety of cell types. Common to most such nanodomains are clustered assemblies of the principal intracellular Ca2+ release channels, ryanodine eceptors (RyRs). JMCs found in cardiac muscle cells have been studied extensively as self-assembled clusters of RyR. While known to form crystalline arrays in vitro, the organization of RyRs in situ within the JMCs has been less clear. The development of single-molecule localization microscopy (SMLM or super-resolution) optical methods have transformed our ability to visualize and accurately quantify the spatial geometries and sizes of RyR clusters. The recent application of the novel DNA-PAINT super-resolution technology has exploited an unprecedented optical resolution of 10–15 nm to visualize the natural arrays of RyRs within JMCs. In this chapter, we review the key insights into the in situ RyR assembly within cardiac nanodomains that have been gained over the last decade with the utility of super-resolution microscopy and the major considerations in interpreting and validating such image data.

To request a preprint version of the chapter, please contact the authors via Researchgate.

Full text of the chapter can be accessed via this direct link.

 

MRC competition funded project available: “Accelerating super-resolution microscopy with machine learning and pattern recognition”

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https://www.findaphd.com/search/ProjectDetails.aspx?PJID=93052

A new 3.5-year PhD opportunity is now open in our group to incorporate Artificial Intelligence and Pattern Recognition towards accelerating one of the most exciting Biomedical imaging technologies available today.

Single molecule localisation microscopy (SMLM) is an invaluable tool for visualising molecular scale interactions and structures which are fundamental to life at the sub-cellular level. Super resolution images are assembled laboriously in SMLM experiments molecule by molecule; therefore it is a time intensive imaging modality which requires up to 90 mins to generate each image. These constraints have been a major barrier to its utility in the Medical Sciences. For example, the slow acquisition speed has made it undesirable for realtime live cell imaging experiments. In the proposed project, we will build a stand-alone programme which can reconstruct the fine spatial features of super-resolution images in a mere fraction of the time needed for recording a full image with a standard protocol. The programme will achieve this by combining existing single molecule localisation algorithms implemented in Python and pattern-determination tools which we have developed to predict the final image iteratively. The software will use an iterative error estimation and image correction algorithm which will feed into a machine learning (ML) platform built-in with Python to inform the process of pattern determination. As the software learns iteratively, to perform pattern determination faster and more robustly, the time required to reconstruct the image using a live stream of primary image data is expected to be abbreviated significantly. This unprecedented level of speed will unlock a wide range of innovative and complex experiments as well as novel technologies which have not been possible thus far. For example, the added speed will enable molecular-scale mapping of rapid cellular events (in the order of seconds) in living disease models.

The successful candidate will work with Dr I.Jayasinghe (www.musclesuperres.com; Twitter @i_jayas), Prof Nikita Gamper (https://www.fbs.leeds.ac.uk/staff/profile.php?tag=Gamper) and Dr Joanna Leng (http://www.joannaleng.com/) who have combined expertise in super-resolution microscopy, cell biology, image analysis, visualisation and ML. Please get in touch now

Showcasing the recent and ongoing DNA-PAINT experiments at the Edinburgh Super Resolution Imaging Consortium Symposium

Isuru attended the second annual Edinburgh Super Resolution Imaging Consortium (ESRIC) symposium, held this year at the Institute of Genetics and Molecular Medicine (IGMM) of the University of Edinburgh (UoE). His talk on the Molecular-scale imaging of ryanodine receptors at both the cell surfaces and interiors with the adaptation of DNA-PAINT was well-received by a range of researchers based in Edinburgh and regionally in Europe.

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Highlights from this meeting included a number of world class investigations led by research fellows and academics in UoE and Heriot Watt University. Of note, were Dr Colin Rickman’s talk on using naturally occurring enzymes as super-resolution imaging probes, Dr Lynn Paterson’s adaptation of optofluidic devices and optical tweezers for developing novel optical tools for cell biology. The plenary speaker was Prof Christophe Zimmer (from Institut Pasteur) who spoke about the adaptation of artificial neuronal networks (a tool called ANNA-PALM) to speed up super-resolution microscopy and demonstrate high throughput imaging of structures such as microtubules, nuclear pore complexes and mitochondria. We now eagerly anticipate his paper on ANNA-PALM out in press very soon.

The conference was organised by Dr Ann Wheeler and colleagues of the ESRIC and showcased their world class line up of microscopy platforms including a state-of-the-art Nikon STORM and SIM instrument and a Leica STED system.

Our research featured on the news of local TV station: ‘Made in Leeds’

On the back of our recent publication in Cell Reports, our research has enjoyed a wide ranging body of TV coverage. This included 20+ online newspapers and science & technology websites. Among this coverage, was a brief recording for the local television station Made in Leeds which was featured on the 6:30pm news on 11/1/2018. 

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Here is the link to a clip where Isuru is explaining the context and the value of the super-resolution microscopy technology in studying both healthy and disease physiology of the heart. Featured in the video, was Miriam during one of her imaging experiments.

Press coverage: “New technique offers ‘perfect window’ to examine causes of heart disease”

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Recent work of researchers from the Universities of Exeter, Leeds and Cambridge has been featured in a number of press reports.

In particular, the Yorkshire Evening Post refer to the recently published paper in Cell Reports in their statement that “A pioneering new technique could boost the understanding of the causes of heart disease, a study suggests.”

Professor Christian Soeller, based at the Living Systems Institute at the University of Exeter expands upon the pioneering nature of this research, stating: “Slightly more than a decade ago nobody thought that we would ever see individual molecules with light, the resolution just seemed insufficient to resolve such fine detail. Since then an astonishing array of new tricks has been devised. In our latest advance, the use of synthetic DNA has been critical – the deep understanding of the chemistry of DNA we have today makes it an enormously versatile tool.”

Dr Isuru Jayasinghe, at the University of Leeds, has provided context to this research in regard to heart failure, having said that: “This new super-resolution microscopy tool gives us the perfect window to visually examine the individual protein changes within heart cells’ molecular machinery which lead to heart failure. At present, none of the treatments or therapies provided to heart failure patients specifically target the signalling stations – nanodomains – within the cell, which the evidence overwhelmingly suggests are a major cause of heart failure. We believe that by visualising these signalling structures at this level of detail using super-resolution microscopy we can help guide investigations into how we can target or repair these molecular machines and thus, in the long term, help patients to overcome heart disease.”

Here at the University of Leeds, Dr Isuru Jayasinghe’s group is utilising multiple super-resolution microscopy techniques to reveal further the fine structural properties of the heart within health and disease.

Read more at: https://www.yorkshireeveningpost.co.uk/news/regional/new-technique-offers-perfect-window-to-examine-causes-of-heart-disease-1-8948322″

See the University of Leeds Science News page  http://www.leeds.ac.uk/news/article/4167/pioneering_technique_could_boost_understanding_of_heart_disease

European Pharmaceutical Review:  https://www.europeanpharmaceuticalreview.com/news/71582/powerful-technique-study-nanodomains/

Medical Xpress: https://medicalxpress.com/news/2018-01-technique-boost-heart-disease.html

EurekAlert: https://www.eurekalert.org/pub_releases/2018-01/uoe-pnt010918.php

 

New paper “True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors” now out on Cell Reports

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Our latest paper together with Christian Soeller’s group is now available with open access. In this paper, we utilise the recently-described DNA-PAINT technique to achieve optical resolutions of ~10 nm as a clear improvement over the more widely used dSTORM super-resolution technique.  With the improved resolution, we were able to visualise and ‘count’ individual proteins which are clustered tightly within intracellular signalling nanodomains in muscle cells of the heart. From this analysis, we learned that the giant ryanodine receptor calcium release channels are organise more loosely and heterogeneously than previously thought. It also appeared that from cluster to cluster, their calcium release properties may be regulated more differentially by varying degrees of co-clustering with regulatory protein junctophilin-2. The study itself was also a demonstration that the enhanced resolution and the new protein ‘counting’ tools are promising new tools for studying molecular-scale biophysics in excitable cells.

The picture above illustrates, with correlative image, the remarkable improvements made by the recent dSTORM and DNA-PAINT techniques over traditional optical microscopy methods like TIRF which were state-of-the-art only ~ 13 years ago.

MRC-funded industrial CASE PhD opportunity advertised

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An exciting PhD project to study the intricate muscle membrane remodelling associated with exercise in elderly people has been advertised. This examines nano/micrometre scale changes in the membrane tubular systems (see picture from our recent paper – Cully et al. (2017) Nature Communications) in skeletal muscle. It is an opportunity to work with our long term skeletal muscle collaborators in Brisbane, clinical collaborators in sunny Devon and autophagy experts in Newcastle with a structured programme to work with industrial partner Badrilla Ltd developing antibody and peptide probes.

See the Opportunities section or FindAPhD.com advert for details. The deadline is the 20/01/2018.

Contact the supervisory team via Dr Jayasinghe.