Biology subjects

Jayasinghe, I.

Publications and source records attributed to Jayasinghe, I..

3 recordsLinked to original sources

bioRxiv2020

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

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.

biophysics↗

bioRxiv2020

Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes

To study the structural basis of pathological remodelling and altered calcium channel functional states in the heart, we sought to re-purpose high-affinity ligands of the cardiac calcium channel, the ryanodine receptor (RyR2), into super-resolution imaging probes. Imperacalcin (IpCa), a scorpion toxin peptide which induces channel sub-conduction states, and DPc10, a synthetic peptide corresponding to a sequence of the RyR2, which replicates arrhythmogenic CPVT functional changes, were used in fluorescent imaging experiments. Isolated adult rat ventricular cardiomyocytes were saponin-permeabilised and incubated with each peptide. IpCa-A546 became sequestered into the mitochondria. This was prevented by treatment of the permeabilised cells with the ionophore FCCP, revealing a striated staining pattern in confocal imaging which had weak colocalisation with RyR2 clusters. Poor specificity (as an RyR2 imaging probe) was confirmed at higher resolution with expansion microscopy (proExM) (~70 nm). DPc10-FITC labelled a striated pattern, which had moderate colocalisation with RyR2 cluster labelling in confocal and proExM. There was also widespread non-target labelling of the Z-discs, intercalated discs, and nuclei, which was unaffected by incubation times or 10 mM caffeine. The inactive peptide mut-DPc10-FITC (which causes no functional effects) displayed a similar labelling pattern. Significant labelling of structures unrelated to RyR2 by both peptide conjugates makes their use as highly specific imaging probes of RyR2 in living isolated cardiomyocytes highly challenging. We investigated the native DPc10 sequence within the RyR2 structure to understand the domain interactions and proposed mechanism of peptide binding. The native DPc10 sequence does not directly interact with another domain, and but is downstream of one such domain interface. The rabbit Arg2475 (equivalent to human Arg2474, mutated in CPVT) in the native sequence is the most accessible portion and most likely location for peptide disturbance, suggesting FITC placement does not impact peptide binding.

cell biology↗

bioRxiv2020

Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

Stochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the off-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at [~] 3-times the speed and [~] 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

biophysics↗