Tracking hemichannels and gap junction dynamics during inflammation through the eyes of correlative imaging at the soft X-ray tomography beamline in the UK’s National Synchrotron facility

Session

Correlative and Multimodal X-ray Microscopy

Authors

Dr Chidinma Okolo (1), Professor Agustin Martinez (2), Dr Maria Harkiolaki (1), Dr Omolola Odunayo (3)

Affiliations

1. Diamond Light Source
2. Universidad de Valparaíso, Valparaíso
3. University of Auckland

Keywords

Gap junctions, hemichannels, connexin43, soft X-ray tomography, structured illumination microscopy

Abstract text

Connexin43 (Cx43) hemichannels have been implicated in several inflammatory diseases affecting various organ systems. Current therapies that inhibit cx43 hemichannels target the blocking of the pore to prevent the pathological release or uptake of ions & signalling molecules. These agents are known to be effective in several disease models. By establishing the mechanism of action of hemichannel blockers, we can better understand the mode of action of current therapies & tailor/repurpose better-targeted therapies for the treatment of cx43-hemichannel related diseases. There is no robust documentation on the impact of inflammation on ultrastructure. Also, there is no native-state super-resolution 3D imaging information on the mechanism of action of cx43 blockage-mediated rescuing of cell morphology and ultrastructure. It became imperative to use ultrastructural changes as an atlas to decipher connexin43 channels behaviour in health and disease (inflammation). The fine cellular phenotypes that needed to be captured in this project meant that diffraction-limited conventional microscopy techniques and sample preparation techniques involving harsh chemical treatments were not suitable for this work. Hence, the need for a platform which combines high resolution localisation fluorescence imaging with high-definition native imaging of the entire cellular landscape (correlative light and X-ray tomography).  

Soft X-ray cryo-tomography (cryoSXT) is a robust 3D cryo-imaging technique that enables cytophysiology research across the length and breadth of the life science. This is because cryoSXT currently offers near-native, high-definition, three-dimensional imaging of the cellular ultrastructure without the need for any artefact-inducing chemical and mechanical processing. CryoSXT delivers high-throughput in-cellulo resolution of up to 25nm in dispersed, adherent, or in-tissue cell populations which are up to 12 microns thick, taking advantage of the inherent absorption of soft X-rays of structured carbon without the requirement for fixation, permeabilization, sectioning, milling, or staining. The method has been paired with other microscopy techniques such as super-resolution fluorescence microscopy, X-ray fluorescence and X-ray absorption spectroscopy to disambiguate features, localise chemical information, track molecular pathways, cellular trafficking and introduce other dimensions beyond the reach of cryoSXT.

B24 is the soft X-ray tomography (SXT) beamline at Diamond Light Source operating at the water window and provides 3D imaging to 25 nm resolution of the cellular landscapes in samples up to 12 μm thick. The beamline offers a fully integrated semi-automated user-friendly correlative imaging platform that encompasses sample preparation and data processing and complements SXT with sample mapping and same-sample 3D super resolution fluorescence. Sample preparation techniques have been greatly standardised in our setting to minimise artefacts and boost the confidence interval when drawing qualitative and quantitative conclusions.

HeLa cells transfected with connexin43-EGFP plasmids were grown on hydrophilized biocompatible grids and driven to an inflammatory state with pro-inflammatory factors- TNFα, IL1β and high glucose levels of 32.5 mM. Thereafter, filamentous Actin, mitochondria and lysosomes were fluorescently labelled in the live cells prior to vitrification by plunge freezing. The cryo structured illumination microscope (cryoSIM) at B24 was used to obtain chemical localisation of signals before the same grids were transferred to the transmission X-ray microscope for cryoSXT data collection. Raw data from cryoSIM were reconstructed with SoftWorX (GE Healthcare) while raw data from cryoSXT were reconstructed through an automated pipeline based on IMOD. Datasets from both microscopes were brought together with minimal nanometre error margin using ec-CLEM.

Results from this study showed that ultrastructural changes in the cytoskeleton, mitochondria and   lysosome could model and predict connexin43 channels behaviour. We also deciphered the curative and preventative impact of the peptide mimetic drug, Xentry-Gap19, used to ameliorate the deleterious impact of inflammation. Taken together, we narrate a story centred on connexin43’s central role in health and disease.

Here, we present the application of a new platform of cryo-imaging in a project that studies connexin43 which is central in the treatment of neurological disorders, cancers, inflammatory disease, diabetic retinopathy, and heart diseases. By capturing key events in this system, we enhance our understanding of the cellular dynamics in inflammation and add the structural dimension to the existing molecular understanding of the processes involved. Our imaging platform is fully commissioned, easily accessible, and ready to serve the wider biomedical community.