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Advanced super-resolution microscopy approaches to observing the membrane remodelling mechanisms in the ischemic heart

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  • Full or part time
    Dr I Jayasinghe
    Dr D Steele
  • Application Deadline
    No more applications being accepted
  • Funded PhD Project (European/UK Students Only)
    Funded PhD Project (European/UK Students Only)

Project Description

Summary

Proteolytic cleavage and mRNA translational suppression are at the heart of the loss of membrane anchoring protein junctophilin-2 (JP2) which occurs almost universally in heart pathologies that lead to heart failure. This PhD project will take on a series of experiments that utilise the next generation of super-resolution microscopy methods, cell culture, “lab on a chip” microfluidic cell perfusion and calcium imaging to visualise the above mechanisms of JP2 loss under ischemic conditions. The chosen candidate will integrate into a world-leading super-resolution microscopy team specialising cardiac muscle cell imaging and a network of multi-disciplinary collaborators in fields which include Molecular Biology, Biophotonics, Biochemistry and Biophysics. Anticipated endpoints of the project include a detailed mechanistic understanding of the structural (particularly membrane topologies) and functional changes in the healthy and ischemic heart, development and refinement of advanced super-resolution microscopy techniques and high impact publications in leading scientific journals.

Background

The rapid and synchronised contraction of muscle cells of the ventricles beat after beat is crucial for the heart’s function as the pump that drives the blood circulation. At the single cell level, this is achieved primarily through a highly organised architecture of plasma membrane invaginations (called t-tubules) which deliver a highly synchronised calcium signal via microscopic arrays of calcium release channels (ryanodine receptors; RyRs) to the cell’s contractile proteins. The correct/healthy structure of t-tubules and RyR arrays are maintained by a number of membrane tethering proteins which are often altered in a range of pathologies that lead to heart failure. In particular, the primary molecular anchor of t-tubules – junctophilin-2 (JP2) is lost in virtually every case of heart failure (Wei et al. 2010). In addition to its role in the formation and growth of t-tubules during development, JP2 expression can counter the severity of the heart’s mechanical impairment during the cardiac pathology.

One of the major challenges in studying t-tubular structures and their intrinsic organisation of proteins such as RyR and JP2 is that they are too small to be visualised with optical microscopy methods. In recent times, super-resolution microscopy has overcome this limitation to reveal an amazing level of detail of the molecular assemblies of these intricate structures (e.g. Baddeley et al 2009, Jayasinghe et al 2012). Now with the next generation of super-resolution imaging methods which include DNA-PAINT (Jungmann et al 2014) and Oligopaint (Beliveau et al 2015) techniques, we are for the first time able to visualise DNA-, RNA- and protein level expression of proteins like JP2 or RyR within living and/or hydrated cells at a resolution paralleling electron microscopy.


Project objectives

1. Establish a cardiac muscle primary culture under hypoxic conditions to simulate the myocardial environment in ischemic events
2. Use DNA-PAINT and Oligopaint super-resolution methods to image:
- JP2 protein and mRNA,
- other proteins that regulate JP2 protein expression
- expression of proteases that can cleave JP2 and
- the 3D structure of t-tubules and dyads
inside heart muscle cells

3. Layer DNA-PAINT experiments with live cell calcium imaging to observe both structure and function within the same cell with the utility of microfluidic (“lab on a chip”) method.

4. Use in vitro biochemical techniques to quantify protein and mRNA targets of interest (e.g. JP2, RyR) to validate the super-resolution image-based measurements

In addition to the above experimental objectives, the candidate will be given domestic and international travel opportunities to carry out collaborative work and present findings at international scientific conferences. The project duration will be 3.5 years and will include a stipend.

Relevance and timeliness

Ischemia is the leading cause of heart failure and the UK’s leading cause of mortality and long term morbidity. This project brings together the latest of the Nobel prize winning (2014) super-resolution microscopy concepts to resolve a molecular pathological mechanism that is at the very heart of this problem. The loss of JP2 has been well documented in heart disease, however this project will be one of the first to develop an experimental understanding of the mechanisms of JP2 loss from both mRNA silencing and proteolytic degredation. Key molecular targets identified in this project can become prime targets for cardioprotective therapies in further work. The project will be hosted in one of the first laboratories in the UK to establish the DNA-PAINT ultra-super-resolution technique and will therefore enable the visualisation of structures and molecular complexes within the heart at a level of detail that has never been possible before.

Funding Notes

The project is fully-funded and will be hosted in the laboratories of both supervisors. The funding is only open to UK/EU students (subject to eligibility); self-funded students are also encouraged to apply. For further details, please contact Dr Jayasinghe ([Email Address Removed]) or Prof Steele ([Email Address Removed]).

References

Baddeley, D., I. D. Jayasinghe, et al. (2009). "Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes." Proc Natl Acad Sci U S A 106(52): 22275-22280.

Beliveau, B. J., A. N. Boettiger, et al. (2015). "Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes." Nat Commun 6: 7147.

Jayasinghe, I. D., D. Baddeley, et al. (2012). "Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes." Biophys J 102(5): L19-21.

Jungmann, R., M. S. Avendano, et al. (2014). "Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT." Nat Methods 11(3): 313-318.

Wei, S., A. Guo, et al. (2010). "T-tubule remodeling during transition from hypertrophy to heart failure." Circ Res 107(4): 520-531.

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