Session 7

Super-resolution expansion microscopy

Professor Markus Sauer, University of Wurzberg, Germany

In the last decade, super-resolution microscopy has evolved as a very powerful method for sub-diffraction resolution fluorescence imaging of cells and structural investigations of cellular organelles. Super-resolution microscopy methods can now provide a spatial resolution that is well below the diffraction limit of light microscopy, enabling invaluable insights into the spatial organization of proteins in biological samples. However, current super-resolution measurements become error-prone below 25 nm. An alternative approach to bypass the diffraction limit and enable “super-resolution imaging” on standard fluorescence microscopes, is the physical expansion of the cellular structure of interest. By linking a protein of interest into a dense, cross-linked network of a swellable polyelectrolyte hydrogel, biological specimens can be physically expanded allowing ~70 nm lateral resolution by confocal laser scanning microscopy. Since its first introduction by Boyden and co-workers in 2015, expansion microscopy (ExM) has shown impressive results including the magnified visualization of pre- or post-expansion labeled proteins and RNAs with fluorescent proteins, antibodies, and oligonucleotides, respectively, in cells, tissues, and human clinical specimen. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using reference structures, neurons and brain slices, we demonstrate that post-labeling Ex-SMLM can be used advantageously for super-resolution imaging. It preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

Imaging the trans-synaptic transfer mechanism of rabies virus.

Dr Vinod Sundaramoorthy, ARC DECRA Research fellow, Deakin University & CSIRO-ACDP

Rabies is a lethal neurotrophic virus which spreads exclusively within interconnected neurons in the host nervous system by transferring across neuronal junctions (synapses). This ability enables the rabies virus to mostly evade immune detection and cause irreversible damage to the nervous system. The trans-synaptic transfer ability of attenuated rabies virus is also exploited beneficially as an anatomical neuronal circuit tracer and as a potential vector system to carry therapeutics to the brain. However, the mechanisms that facilitate the transfer of rabies virus across the synapse is unknown. While the envelope protein (glycoprotein) of rabies virus is responsible for this trans-synaptic transmission, the specific neuronal receptors and associated ultrastructural changes occurring at the neuronal synapse to facilitate viral transfer remains unknown.

In this study, we employed advanced confocal and electron microscopy imaging techniques in high-containment PC3 laboratories to study trans-synaptic transfer of rabies virus in neurons. Using ex-vivo neuronal models, we performed ultrastructural investigation of rabies virus transfer between neurons in live and fixed cultures. In these studies, we identified novel trans-synaptic transfer mechanisms utilised by highly-neuroinvasive and low-neuroinvasive rabies strains in neurons. We also identified novel abilities of rabies virus glycoprotein to efficiently control and modify synaptic architecture in neurons to enable virus transfer. These studies present new information about how rabies spreads through the nervous system, generating valuable knowledge to develop future strategies for rabies treatment and to design next-generation rabies-derived vector systems for brain research and drug delivery.

Super-resolving the nanoscale dynamics of Botulinum Neurotoxin Type-A intoxication in hippocampal neurons

Dr Merja Joensuu, ARC DECRA Research Fellow, Queensland Brain Institute, The University of Queensland

Co-authors
Dr Vanessa Lanoue, Queensland Brain Institute, The University of Queensland
Dr Alisa Blum, Queensland Brain Institute, The University of Queensland
Dr Stefan Mahrhold, Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany
Miss Nadja Krez, Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany
A/Prof Giuseppe Ballistreri, University of Helsinki
Dr Andreas Rummel, Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany
Prof Frederic Meunier, Queensland Brain Institute, The University of Queensland

Neuronal communication is encoded by neurotransmitters stored in synaptic vesicles (SVs) that undergo Ca2+-dependent fusion with the presynaptic plasma membrane (PM) upon stimulation, after which the SVs are rapidly reformed through endocytosis. Neurotoxins, such as Botulinum Neurotoxin Type-A (BoNT/A), the most potent toxin known, hijacks this endocytic pathway to be internalized into nerve terminals as part of their intoxication strategy to incapacitate neuronal communication. According to the long-standing dual-receptor hypothesis, BoNT/A first binds to a specific ganglioside (such as GT1b) on the plasma membrane, and then subsequently to an intraluminal epitope of plasma membrane-stranded synaptic vesicle protein 2 (SV2), thereby launching receptor-mediated endocytosis. Following translocation from SVs into the cytoplasm, the toxin’s light chain proteolytic activity then cleaves SNAP-25, a SNARE protein responsible for mediating fusion of SVs with the plasma membrane, leading to neurotransmission block and flaccid paralysis. I will discuss our recent discoveries that challenge the current receptor hypothesis, and introduce a novel hypothesis based on a coincidental engagement of the toxin to both ganglioside and SV2 within nanoclusters that controls the endocytic uptake and sorting of the BoNT/A in primary hippocampal neurons. Using super-resolution and confocal imaging, and electron microscopy, we studied the nanoscale dynamics and mechanisms by which BoNT/A is selectively targetted into SVs. Genetic inactivation of the BoNT/A’s GT1b and SV2 binding sites perturbed PM binding and clustering of the toxins, and the subsequent entry, sorting and retrograde trafficking, indicating that coincidental engagement of BoNT/A with both of the receptors is required for correct entry of the toxin. Single-molecule imaging uncovers an unprecedented dynamic view of the cascade of nanoscale events underpinning BoNT/A intoxication.

SRRF ‘n’ TIRF - Simultaneous spatiotemporal super-resolution and multiparametric fluorescence microscopy

Professor Thorsten Wohland, National University of Singapore

The full description of biological systems requires the measurement of structure and dynamics. The quality of data depends on the signal to noise ratio and thus on the photons detected. However, good spatial resolution requires small pixels and thus longer exposure times for high photon counts. And good dynamics resolution requires fast measurements and thus larger pixels to obtain the required signal to noise ratio. Attempts in the past to combine the two, had limitations on instrumentation or labels. Here we combine computational super-resolution with imaging fluorescence correlation spectroscopy to obtain high spatiotemporal resolution with standard instrumentation and fluorescent proteins.