A correlative super-resolution protocol to map the origins of endoplasmic reticulum calcium release events in dorsal root ganglion neurones

Session

Poster Session Two

Authors

Dr Miriam Hurley (1), Dr Shihab Shah (1), Dr Thomas Sheard (2), Dr Hannah Kirton (1), Prof Derek Steele (1), Prof Nikita Gamper (1), Dr Izzy Jayasinghe (2)

Affiliations

1. University of Leeds
2. University of Sheffield

Keywords

single molecule localisation microscopy; correlative microscopy; expansion microscopy; calcium; ryanodine receptor; dorsal root ganglion

Abstract text

Single molecule localisation microscopy (SMLM) is a common tool used to resolve the nanoscale patterns of proteins. However, nanometre-scale cellular information is often unaccompanied by functional information, in part due to the requirement for cells to undergo fixation and permeabilisation or the incompatibility across image acquisition timescales. 

 

In excitable cell types, calcium (Ca2+) acts as a ubiquitous intracellular second messenger. Multiple Ca2+ handling proteins are organised within nanoscale signalling domains to aid the coordinated release of Ca2+ from the endoplasmic reticulum (ER). Within dorsal root ganglion neurones (DRGs), the release of Ca2+ is mediated by the ryanodine receptor (RyR) and inositol 1,4,5-triphosphate receptor (IP3R). Over the past decade, visualisation of RyR organisation into clusters or arrays has advanced [1]. However, adoption of SMLM technology has not extended to RyRs in DRG. Currently, within DRG neurones, confocal images suggest a clustered sub-plasmalemmal organisation of RyR [2], whilst STORM images visualise nanoscale clusters of IP3R [3]. 

 

We have adapted our previously developed correlative imaging protocol [4] to spatially correlate the two-dimensional (2D) total internal reflection fluorescence (TIRF) live-cell imaging of Ca2+ release from the ER with the subsequent dSTORM imaging of RyR and IP3R sub-plasmalemmal nanoscale patterns in DRG neurones. DRG somata were isolated from neonatal rats (6-10 days) and co-cultured with satellite glial cells onto a glass dish with gridded bottom. Cells were loaded with the cytoplasmic Ca2+ indicator Fluo-4 AM before being exposed to a 5 mM extracellular Ca2+ environment. Transient and localised Ca2+ release events from the ER were visualised in a 2D time series, with the specific grid coordinates of cells recorded. Cells were then fixed in situ and labelled with anti-RyR or anti-IP3R antibodies for dSTORM acquisition. 

 

Using a semi-automated detection system [1], discretisation of individual RyR and IP3R channels enabled spatial statistics of each channel to be extracted and their centroid to be detected. Within the same cellular region, Ca2+ release events were characterised from the application of a 2D-Guassian filter detection protocol to identify the centroid of each spatial footprint (0.5-2.0 mm in width). Alignment of the respective RyR or IP3R channels with the location of Ca2+ release events was by a two-step process, through the establishment of user-driven ‘primary alignment vectors’ followed by cross-correlation in Fourier space. When overlaid and aligned, spatial statistics could be undertaken in regard to the number of RyRs or IP3R detected underneath the footprint of each Ca2+ release event. 

 

Overlay of functional and structural data revealed coinciding clusters of Ca2+ handling proteins with Ca2+ release events, with a spatial heterogeneity in spontaneous Ca2+ activity. Of the Ca2+ release events sampled, over half had RyR puncta underneath their footprint (19.4±26.6 mean±SEM), compared to a two-fold increase in IP3R puncta (34.8±41.79 mean±SEM). In addition, correlation between spark mass and IP3R localisation was weaker compared to RyR organisation when the mass of a Ca2+ release event was <1 A.U., with higher counts of IP3R consistently observed when the mass of a Ca2+ release event was >10 A.U. Combined, this suggests that RyR is the primary generator of Ca2+ release events within DRGs, with IP3R acting as a primer or amplifier of RyR-mediated spontaneous Ca2+ release. 

Within DRG somata that did not undergo the above correlative imaging protocol, nanoscale distribution of IP3R and RyR was observed using a 10x enhanced expansion microscopy (EExM) protocol [5]. Images were acquired upon an inverted LSM880 (Carl Zeiss, Jena) in Airyscan mode using a Plan-Apochromat 40x 1.3 NA objective that enabled an in-plane resolution of 15 nm. The distribution of IP3R was spatially-heterogeneous, with a punctate morphology, whilst RyR pattern had noticeable clustering. These morphologies were a qualitative and quantitative reproduction of dSTORM images previously acquired. 

Here we have developed an experimental protocol that enabled us to probe the local structure-function relationship in a primary cell type. This protocol has enabled the correlative imaging of sub-plasmalemmal second-messenger signals, such as Ca2+ release from the ER, with the local spatial organisation of proteins at a nanometre-scale. As such, our observations have revealed distinct RyR and IP3R patterns between the ER and surface plasmalemma that encode the position and quantity of calcium release. 

References

References:

[1] Jayasinghe, I. et al. (2018) True molecular scale visualization of variable clustering properties of Ryanodine Receptors. Cell Reports, 22, 557-567. 

[2] Ouyang, K. et al. (2005) Ca2+ sparks and secretion in Dorsal Root Ganglion Neurons. Proc Natl Acad Sci USA, 102, 12259-12264. 

[3] Shah, S. et al. (2020) Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels. Sci Signal, 13, 629. 

[4] Hurley, M.E. et al. (2021) A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types. Methods, 193, 27-37.

[5] Sheard, T.M.D. et al. (2019) Three-dimensional and chemical mapping of intracellular signalling nanodomains in health and disease with enhanced expansion microscopy. ACS Nano, 13, 2143-2157.