Chemical communication is underpinned by the fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane at active zones. With the advent of super-resolution microscopy, the door ...is now opened to unravel the dynamic remodeling of synapses underpinning learning and memory. Imaging proteins with conventional light microscopy cannot provide submicron information vital to determining the nanoscale organization of the synapse. We will first review the current super-resolution microscopy techniques available to investigate the localization and movement of synaptic proteins and how they have been applied to visualize the synapse. We discuss the new techniques and analytical approaches have provided comprehensive insights into synaptic organization in various model systems. Finally, this review provides a brief update on how these super-resolution techniques and analyses have opened the way to a much greater understanding of the synapse, the fusion and compensatory endocytosis machinery.
•Super-resolution microscopy approaches are key to uncovering the dynamic organization of the synapse.•Single-particle tracking has allowed us to uncover a more detailed mechanism of synaptic plasticity.•Advanced analytical approaches have provided insights into the transient nanoclustering of the remodelling synapse.
Single-molecule localization microscopy techniques are emerging as vital tools to unravel the nanoscale world of living cells by understanding the spatiotemporal organization of protein clusters at ...the nanometer scale. Current analyses define spatial nanoclusters based on detections but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in video games to detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to determine the overlap of the bounding boxes of individual molecular trajectories to establish membership in nanoclusters. Extending the spatial indexing into the time dimension allows the resolution of spatial nanoclusters into multiple spatiotemporal clusters. Using spatiotemporal indexing, we found that syntaxin1a and Munc18-1 molecules transiently cluster in hotspots, offering insights into the dynamics of neuroexocytosis. Nanoscale spatiotemporal indexing clustering (NASTIC) has been implemented as a free and open-source Python graphic user interface.
Protein kinases (PKs) are proteins at the core of cellular signalling and are thereby responsible for most cellular physiological processes and their regulations. As for all intracellular proteins, ...PKs are subjected to Brownian thermal energy that tends to homogenise their distribution throughout the volume of the cell. To access their substrates and perform their critical functions, PK localisation is therefore tightly regulated in space and time, relying upon a range of clustering mechanisms. These include post-translational modifications, protein-protein and protein-lipid interactions, as well as liquid-liquid phase separation, allowing spatial restriction and ultimately regulating access to their substrates. In this review, we will focus on key mechanisms mediating PK nanoclustering in physiological and pathophysiological processes. We propose that PK nanoclusters act as a cellular quantal unit of signalling output capable of integration and regulation in space and time. We will specifically outline the various super-resolution microscopy approaches currently used to elucidate the composition and mechanisms driving PK nanoscale clustering and explore the pathological consequences of altered kinase clustering in the context of neurodegenerative disorders, inflammation, and cancer.
Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the ...presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2a
), but not the tetramerization-deficient mutant K337Q (Syn2a
), fully rescued these effects. Single-particle tracking revealed that Syn2a
mEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.
Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at ...discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.
Abstract
Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the ...reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.
None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. ...Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β₂-adrenoreceptor (β₂-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β₂-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β₂-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.
Propofol is the most commonly used general anesthetic in humans. Our understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is ...unknown whether other neural mechanisms are involved in general anesthesia. Here, we demonstrate that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, we show that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. Our results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.
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•Propofol impairs presynaptic release of neurotransmitters•Propofol and etomidate restrict syntaxin1A mobility on presynaptic membranes•Non-anesthetic analogs of propofol increase syntaxin1A mobility•A propofol target emerges from an interaction between syntaxin1A and SNAP-25
Bademosi et al. use single-molecule imaging microscopy to understand how general anesthetics might affect presynaptic release mechanisms. They find that a clinically relevant concentration of propofol targets the presynaptic release machinery by specifically restricting syntaxin1A mobility on the plasma membrane. This suggests an alternate target process for these drugs.
Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, we ...hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. Here we show that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor, transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes.
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