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bioRxiv · 10.1101/2020.09.29.319400

A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types

Hurley, M. E.·Sheard, T. M. D.·Norman, R.·Kirton, H. M.·Shah, S. S.·Pervolaraki, E.·Yang, Z.·Gamper, N.·White, E.·Steele, D.·Jayasinghe, I.
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Abstract

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

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Hurley, M. E., Sheard, T. M. D., Norman, R., Kirton, H. M., Shah, S. S., Pervolaraki, E., Yang, Z., Gamper, N., White, E., Steele, D., Jayasinghe, I.. 2020-09-30. A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types. https://doi.org/10.1101/2020.09.29.319400

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