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Numerous models have been developed to account for the complex properties of the random walks of biomolecules. However, when analysing experimental data, conditions are rarely met to ensure model identification. The dynamics may simultaneously be influenced by spatial and temporal heterogeneities of the environment, out-of-equilibrium fluxes and conformal changes of the tracked molecules. Recorded trajectories are often too short to reliably discern such multi-scale dynamics, which precludes unambiguous assessment of the type of random walk and its parameters. Furthermore, the motion of biomolecules may not be well described by a single, canonical random walk model. Here, we develop a two-step statistical testing scheme for comparing biomolecule dynamics observed in different experimental conditions without having to identify or make strong prior assumptions about the model generating the recorded random walks. We first train a graph neural network to perform simulation-based inference and thus learn a rich summary statistics vector describing individual trajectories. We then compare trajectories obtained in different biological conditions using a non-parametric maximum mean discrepancy (MMD) statistical test on their so-obtained summary statistics. This procedure allows us to characterise sets of random walks regardless of their generating models, without resorting to model-specific physical quantities or estimators. We first validate the relevance of our approach on numerically simulated trajectories. This demonstrates both the statistical power of the MMD test and the descriptive power of the learnt summary statistics compared to estimates of physical quantities. We then illustrate the ability of our framework to detect changes in α -synuclein dynamics at synapses in cultured cortical neurons, in response to membrane depolarisation, and show that detected differences are largely driven by increased protein mobility in the depolarised state, in agreement with previous findings. The method provides a means of interpreting the differences it detects in terms of single trajectory characteristics. Finally, we emphasise the interest of performing various comparisons to probe the heterogeneity of experimentally acquired datasets at different levels of granularity (e.g., biological replicates, fields of view, and organelles).

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion.

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

The application of super-resolution optical microscopy to investigating synaptic structures has revealed a highly heterogeneous and variable intra-synaptic organization. Dense subsynaptic protein assemblies named subsynaptic domains or SSDs have been proposed as structural units that regulate the efficacy of neuronal transmission. However, an in-depth characterization of SSDs has been hampered by technical limitations of super-resolution microscopy of synapses, namely the stochasticity of the signals during the imaging procedures and the variability of the synaptic structures. Here, we synthetize the available evidence for the existence of SSDs at central synapses, as well as the possible functional relevance of SSDs. In particular, we discuss the possible regulation of co-transmission at mixed inhibitory synapses as a consequence of the subsynaptic distribution of glycine receptors (GlyRs) and GABA receptors (GABA Rs). Super-resolution imaging strategies bypass the resolution limit of conventional optical microscopy and have given new insights into the distribution of proteins at synapses in the central nervous system. Neurotransmitter receptors and scaffold proteins appear to occupy specialized locations within synapses that we refer to as subsynaptic domains or SSDs. Interestingly, these SSDs are highly dynamic and their formation seems to be related to the remodeling of synapses during synaptic plasticity. It was also shown that SSDs of pre-and post-synaptic proteins are aligned in so-called nanocolumns, highlighting the role of SSDs in the regulation of synaptic transmission. Despite recent advances, however, the detection of SSDs with super-resolution microscopy remains difficult due to the inherent technical limitations of these approaches that are discussed in this review article.

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans -SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer. The surface area of neurons increases rapidly during neurite extension. Galli and colleagues show that the endoplasmic reticulum (ER)-resident SNARE protein Sec22b bridges the ER and plasma membrane during this process and contributes to plasma membrane expansion, but does not promote membrane fusion.

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far, the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs µm -2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains, and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

Single-molecule localization microscopy (SMLM) enables crucial insights into cellular structures and processes to be revealed at the single-molecule level. However, SMLM is often hampered by limited temporal resolution and the fixed frame rate of the acquisition. Here we present a new approach to SMLM data acquisition and processing based on an affordable event-based sensor. This type of sensor reacts to changes in light intensity, rather than integrating photons during the exposure time of each frame. Each pixel works independently and returns a signal only when an intensity change is detected. Compared with video acquisition using traditional electron-multiplying charge-coupled device or scientific complementary metal–oxide–semiconductor cameras, the event-based sensor provides higher temporal resolution and throughput on the positions of blinking molecules. We demonstrate event-based SMLM super-resolution imaging on biological samples with spatial resolution on a par with the performance of electron-multiplying charge-coupled device or scientific complementary metal–oxide–semiconductor cameras, while registering only the on and off switching of blinking molecules. We use event-based SMLM to perform very dense single-molecule imaging, where frame-based cameras experience major limitations.Event-based sensors enable super-resolution single-molecule localization microscopy with comparable quality and resolution to traditional scientific cameras, while also overcoming the limitations of high-density imaging.

This study shows that, although AMPA-type glutamate receptors are trafficked to dendrites through the normal Golgi secretory pathway, NMDA receptors bypass the somatic Golgi apparatus and instead move from the endoplasmic reticulum in endoplasmic reticulum–like vesicles to Golgi 'outposts' located in the dendrites. Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β‐subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor–gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR–gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β‐subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity. Protein kinase C‐mediated phosphorylation of the glycine receptor‐β (GlyRβ) subunit reduces GlyR affinity for the synaptic scaffold protein gephyrin. This leads to increased GlyR diffusion in the plasma membrane and reduced GlyR accumulation at inhibitory synapses.