Trans synaptic assemblies link synaptic vesicles and neuroreceptors

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling pathways that require a precise protein organization, such as the previously reported nanodomain colocalization of pre and postsynaptic proteins. Here, we used cryo electron tomography to visualize synaptic complexes together with their native environment comprising interacting proteins and lipids on a 2 to 4 nm scale. Using template free detection and classification, we showed that tripartite trans synaptic assemblies subcolumns link synaptic vesicles to postsynaptic receptors and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in plasma membrane. These data support the hypothesis that synaptic function is carried by precisely organized trans synaptic units. It provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their functio

Synucleins Have Multiple Effects on Presynaptic Architecture

Synucleins ( a , b , g -synuclein) are abundant presynaptic proteins, with a -synuclein linked to the pathogenesis of Parkinson’s disease. Vargas et al. investigate the effects of deleting synucleins and overexpressing mutated a -synuclein on synapse architecture using electron microscopy. They find that synucleins regulate presynaptic terminal size and synaptic vesicle distribution

MAN1 Restricts BMP Signaling During Synaptic Growth in Drosophila

Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. Here, we show that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, we propose that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs

Hierarchical detection and analysis of macromolecular complexes in cryo-electron tomograms using Pyto software

Molecular complexes, arguably the basic units carrying cellular function, can be visualized directly in their native environment by cryo-electron tomography. Here we describe a procedure for the detection of small, pleomorphic membrane-bound molecular complexes in cryo-tomograms by a hierarchical connectivity segmentation. Validation on phantom and real data showed above 90% true positive rates. This segmentation procedure is implemented in the Pyto software package, together with methods for quantitative characterization and classification of complexes detected by our segmentation procedure and for statistical analysis between experimental conditions. Therefore, the methods presented provide a means for the detection and quantitative interpretation of structures captured in cryo-electron tomograms, as well as for the elucidation of their cellular function. (C) 2016 Published by Elsevier Inc

Aging Drosophila melanogaster Display Altered Pre- and Postsynaptic Ultrastructure at Adult Neuromuscular Junctions

Although age-related changes in synaptic plasticity are an important focus within neuroscience, little is known about ultrastructural changes of synaptic morphology during aging. Here we report how aging affects synaptic ultrastructure by using fluorescence and electron microscopy at the adult Drosophila neuromuscular junction (NMJ) of ventral abdominal muscles. Mainly four striking morphological changes of aging NMJs were revealed. 1) Bouton size increases with proportionally rising number of active zones (AZs). 2) Synaptic vesicle density at AZs is increased in old flies. 3) Late endosomes, cisternae, and multivesicular bodies accumulate in the presynaptic terminal, and vesicles accumulate between membranes of the terminal bouton and the subsynaptic reticulum. 4) The electron-dense pre- and postsynaptic apposition is expanded in aging NMJs, which is accompanied by an expansion of the postsynaptic glutamate receptor fields. These findings suggest that aging is possibly accompanied by impaired synaptic vesicle release and recycling and a potentially compensatory expansion of AZs and postsynaptic densities. (C) 2015 Wiley Periodicals, Inc

Electron cryotomography of vitrified cells with a Volta phase plate

Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1-3 nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (similar to 400 nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-beta-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow. (C) 2015 Elsevier Inc. All rights reserved

Approaches for classification of pipes and tubes in georadargrams

Ground penetrating radar is widely used for the detection of buried pipes and cables. These objects typically appear as hyperbolas in the georadargrams. After detection of such a feature in a section, the question arises whether further information about the material and geometry of the subsurface object can be extracted from a single scan or profile of the reflected signal. Classification attempts based on the reflected amplitude and shape of the wavelet alone seem to be of minor practical importance, because local soil variations can be large and unpredictable, and the reproducibility of measurements, because of antenna coupling or limitations of radar equipment, is not adequate. Thus, we have checked different approaches for a rough classification of pipes and cables, which can reveal diameter (eccentricity migration), material (polarization analysis) and information about the soil structure (texture analysis)

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation