Modulating Vesicle Priming Reveals that Vesicle Immobilization Is Necessary but not Sufficient for Fusion-Competence

In neurons and neuroendocrine cells, docked vesicles need to undergo priming to become fusion competent. Priming is a multi-step process that was shown to be associated with vesicle immobilization. However, it is not known whether vesicle immobilization is sufficient to acquire complete fusion competence. To extend our understanding of the physical manifestation of vesicle priming, we took advantage of tomosyn, a SNARE-related protein that specifically inhibits vesicle priming, and measured its effect on vesicle dynamics in live chromaffin cells using total internal reflection fluorescence microscopy. We show here that while in control cells vesicles undergo immobilization before fusion, vesicle immobilization is attenuated in tomosyn overexpressing cells. This in turn increases the turnover rate of vesicles near the membrane and attenuates the fusion of newcomer vesicles. Moreover, the release probability of immobile vesicles in tomosyn cells is significantly reduced, suggesting that immobilization is an early and necessary step in priming but is insufficient, as further molecular processes are needed to acquire complete fusion competence. Using tomosyn as a molecular tool we provide a mechanistic link between functional docking and priming and suggest that functional docking is the first step in vesicle priming, followed by molecular modifications that do not translate into changes in vesicle mobility

Imaging the assembly and disassembly kinetics of cis-SNARE complexes on native plasma membranes

AbstractMild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (α-SNAP) concentrations

Tomosyn Expression Pattern in the Mouse Hippocampus Suggests Both Presynaptic and Postsynaptic Functions

The protein tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of tomosyn1 and tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of tomosyn in neurotransmitter release, tomosyn2 might have a postsynaptic function, and place tomosyn as a more general regulator of synaptic transmission and plasticity

Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters

Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization

Effect of SARS-CoV-2 proteins on vascular permeability.

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein–protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While vali-dating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-1

Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson's disease model.

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases