Quantifying molecular aggregation by super resolution microscopy within an excitatory synapse from mouse hippocampal neurons.
Ontology highlight
ABSTRACT:
SUBMITTER: Kedia S
PROVIDER: S-EPMC8076707 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Synucleinopathies are characterized by the accumulation of insoluble ?-synuclein (?Syn). To test whether ?Syn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing ?Syn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human ?Syn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of ?Syn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of ?Syn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of ?Syn pathology within 1 d. Moreover, by direct addition of ?Syn PFFs into culture medium, the formation of misfolded ?Syn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with ?Syn PFFs. Our data indicate that intraneuronal ?Syn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein ?-synuclein (?Syn) aggregates to form intraneuronal inclusions. To understand how and to what extent ?Syn aggregates modulate synaptic activity before neuron loss, we demonstrate that ?Syn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of ?Syn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of ?Syn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.
| S-EPMC6595958 | biostudies-literature
Project description:The ubiquitously expressed SUMO proteins regulate a plethora of cellular pathways and processes. While they have a predominantly nuclear localization, extranuclear roles of SUMO isoforms at the synapse have also been described, making SUMOylation one of the major post-translational regulators of nerve functions. These findings have however recently been challenged, at least for SUMO1, by the analysis of knock-in mice expressing His6-HA-SUMO1, where the authors failed to detect the protein at the synapse. In the ongoing dispute, the subcellular distribution in neurons of SUMO2/3 and of the E2 SUMO ligase Ubc9 has not been examined. To investigate whether SUMO proteins do or do not localize at the synapse, we studied their localization in hippocampal primary neurons by super resolution microscopy. We found that SUMO1, SUMO2/3, and Ubc9 are primarily nuclear proteins, which also colocalize partially with pre- and post-synaptic markers such as synaptophysin and PSD95.
| S-EPMC6844275 | biostudies-literature
Project description:Fused in Sarcoma (FUS) is a multifunctional RNA-/DNA-binding protein, which is involved in the pathogenesis of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A common hallmark of these disorders is the abnormal accumulation of mutated FUS protein in the cytoplasm. Under normal conditions FUS is confined to the nuclear compartment, in neurons, however, additional somatodendritic localization can be observed. In this study, we carefully analyzed the subcellular localization of endogenous FUS at synaptic sites of hippocampal neurons which are among the most affected cell types in FTD with FUS pathology. We could confirm a strong nuclear localization of FUS as well as its prominent and widespread neuronal expression throughout the adult and developing rat brain, particularly in the hippocampus, the cerebellum and the outer layers of the cortex. Intriguingly, FUS was also consistently observed at synaptic sites as detected by neuronal subcellular fractionation as well as by immunolabeling. To define a pre- and/or postsynaptic localization of FUS, we employed super-resolution fluorescence localization microscopy. FUS was found to be localized within the axon terminal in close proximity to the presynaptic vesicle protein Synaptophysin1 and adjacent to the active zone protein Bassoon, but well separated from the postsynaptic protein PSD-95. Having shown the presynaptic localization of FUS in the nervous system, a novel extranuclear role of FUS at neuronal contact sites has to be considered. Since there is growing evidence that local presynaptic translation might also be an important mechanism for plasticity, FUS - like the fragile X mental retardation protein FMRP - might act as one of the presynaptic RNA-binding proteins regulating this machinery. Our observation of presynaptic FUS should foster further investigations to determine its role in neurodegenerative diseases such as ALS and FTD.
| S-EPMC4709451 | biostudies-literature
Project description:Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.
| S-EPMC10643044 | biostudies-literature
Project description:The resolution and accuracy of single-molecule localization microscopes (SMLMs) are routinely benchmarked using simulated data, calibration rulers, or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of reconstructed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.
| S-EPMC7732856 | biostudies-literature
Project description:We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.
| S-EPMC5665986 | biostudies-literature
Project description:During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.
| S-EPMC7614930 | biostudies-literature
Project description:Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low-affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin-positive (PV) interneurons. Nrg3 and ErbB4 are located pre- and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short-term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4+ interneurons and affects short-term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non-neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons.
| S-EPMC6120667 | biostudies-literature
Project description:Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low-affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin-positive (PV) interneurons. Nrg3 and ErbB4 are located pre- and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short-term plasticity and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4+ interneurons and affects short-term plasticity. Nrg3-mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non-neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons.
| S-SCDT-EMBOJ-2017-98858 | biostudies-other
Project description:Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.
| S-EPMC4936537 | biostudies-literature