Munc13- and SNAP25-dependent molecular bridges play a key role in synaptic vesicle priming.

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N-ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.

Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Computational methods for ultrastructural analysis of synaptic complexes.

Electron microscopy (EM) provided fundamental insights about the ultrastructure of neuronal synapses. The large amount of information present in the contemporary EM datasets precludes a thorough assessment by visual inspection alone, thus requiring computational methods for the analysis of the data. Here, I review image processing software methods ranging from membrane tracing in large volume datasets to high resolution structures of synaptic complexes. Particular attention is payed to molecular level analysis provided by recent cryo-electron microscopy and tomography methods.

Neurons as a model system for cryo-electron tomography.

Cryo-electron tomography (Cryo-ET) provides unique opportunities to image cellular components at high resolution in their native state and environment. While many different cell types were investigated by cryo-ET, here we review application to neurons. We show that neurons are a versatile system that can be used to investigate general cellular components such as the cytoskeleton and membrane-bound organelles, in addition to neuron-specific processes such as synaptic transmission. Furthermore, the synapse provides a rich environment for the development of cryo-ET image processing tools suitable to elucidate the functional and spatial organization of compositionally and morphologically heterogeneous macromolecular complexes involved in biochemical signaling cascades, within their native, crowded cellular environments.

Statistical spatial analysis for cryo-electron tomography.

Cryo-electron tomography (cryo-ET) is uniquely suited to precisely localize macromolecular complexes in situ, that is in a close-to-native state within their cellular compartments, in three-dimensions at high resolution. Point pattern analysis (PPA) allows quantitative characterization of the spatial organization of particles. However, current implementations of PPA functions are not suitable for applications to cryo-ET data because they do not consider the real, typically irregular 3D shape of cellular compartments and molecular complexes. Here, we designed and implemented first and the second-order, uni- and bivariate PPA functions in a Python package for statistical spatial analysis of particles located in three dimensional regions of arbitrary shape, such as those encountered in cellular cryo-ET imaging (PyOrg). To validate the implemented functions, we applied them to specially designed synthetic datasets. This allowed us to find the algorithmic solutions that provide the best accuracy and computational performance, and to evaluate the precision of the implemented functions. Applications to experimental data showed that despite the higher computational demand, the use of the second-order functions is advantageous to the first-order ones, because they allow characterization of the particle organization and statistical inference over a range of distance scales, as well as the comparative analysis between experimental groups comprising multiple tomograms. Altogether, PyOrg is a versatile, precise, and efficient open-source software for reliable quantitative characterization of macromolecular organization within cellular compartments imaged in situ by cryo-ET, as well as to other 3D imaging systems where real-size particles are located within regions possessing complex geometry.

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 function.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.

Molecular architecture of the presynaptic terminal.

Neurotransmitter release at the presynaptic terminal is one of the fundamental processes in neuronal communication. It is a complex process comprising signaling pathways that exert a precise spatio-temporal coordination to prepare and bring synaptic vesicles to exocytosis. While many molecular components involved have been identified, their direct observation at different stages of the neurotransmitter release is lacking. Three-dimensional imaging by electron tomography provided remarkable views of the synaptic vesicles and the cytomatrix. Imaging fully hydrated, vitrified samples allowed a direct visualization, precise localization and a quantitative characterization of pleomorphic synaptic vesicle-bound complexes in situ, as well as the elucidation of their function in the neurotransmitter release.

Multimodal Synchrotron Radiation Microscopy of Intact Astrocytes from the hSOD1 G93A Rat Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the most common adult onset neurodegenerative disorder affecting motor neurons. Disruptions in metal ion homeostasis have been described in association with ALS, but the pathological mechanisms are still poorly understood. One of the familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1). In this study, we employed orthogonal cellular synchrotron radiation based spectro-microscopies to investigate the astrocytes of an ALS animal model: the rat hSOD1 G93A that overexpresses human mutated SOD1, which is known to increase the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Specifically, we applied soft X-ray transmission tomography and hard X-ray fluorescence microscopy in situ, Fourier transform infrared spectro-microscopy to detect and analyze aggregates, as well as to determine the alterations in the cellular ultrastructure and the elemental and the organic composition of ALS model astrocytes with respect to the control astrocytes isolated from nontransgenic littermates (NTg). The present study demonstrates that large aggregates in the form of multivesicular inclusions form exclusively in the ALS model astrocytes and not in the NTg counterpart. Furthermore, the number of mitochondria, the cellular copper concentration, and the amount of antiparallel ß-sheet structures were significantly changed within the cells of the ALS model as well as the lipid localization and composition. Also, our data indicate that choline was decreased in the ALS model astrocytes, which could explain their higher sensitivity to oxidative stress that we observed. These results show that the hG93A SOD1 mutation causes metabolic and ultrastructural cellular changes and point to a link between an increased copper concentration and aggregation: the most probable that the aggregation of G93A hSOD1 may perturb its binding to Cu, thus directly or indirectly affecting Cu homeostasis.

Pleomorphic linkers as ubiquitous structural organizers of vesicles in axons.

Many cellular processes depend on a precise structural organization of molecular components. Here, we established that neurons grown in culture provide a suitable system for in situ structural investigations of cellular structures by cryo-electron tomography, a method that allows high resolution, three-dimensional imaging of fully hydrated, vitrified cellular samples. A higher level of detail of cellular components present in our images allowed us to quantitatively characterize presynaptic and cytoskeletal organization, as well as structures involved in axonal transport and endocytosis. In this way we provide a structural framework into which information from other methods need to fit. Importantly, we show that short pleomorphic linkers (tethers and connectors) extensively interconnect different types of spherical vesicles and other lipid membranes in neurons imaged in a close-to-native state. These linkers likely serve to organize and precisely position vesicles involved in endocytosis, axonal transport and synaptic release. Hence, structural interactions via short linkers may serve as ubiquitous vesicle organizers in neuronal cells.

Synucleins Have Multiple Effects on Presynaptic Architecture.

Synucleins (α, ß, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αßγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous findings that these PD mutations decrease neurotransmission. Collectively, our results suggest that synucleins are important orchestrators of presynaptic terminal topography.

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.

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.

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.

Topographic Mapping of the Synaptic Cleft into Adhesive Nanodomains.

The cleft is an integral part of synapses, yet its macromolecular organization remains unclear. We show here that the cleft of excitatory synapses exhibits a distinct density profile as measured by cryoelectron tomography (cryo-ET). Aiming for molecular insights, we analyzed the synapse-organizing proteins Synaptic Cell Adhesion Molecule 1 (SynCAM 1) and EphB2. Cryo-ET of SynCAM 1 knockout and overexpressor synapses showed that this immunoglobulin protein shapes the cleft's edge. SynCAM 1 delineates the postsynaptic perimeter as determined by immunoelectron microscopy and super-resolution imaging. In contrast, the EphB2 receptor tyrosine kinase is enriched deeper within the postsynaptic area. Unexpectedly, SynCAM 1 can form ensembles proximal to postsynaptic densities, and synapses containing these ensembles were larger. Postsynaptic SynCAM 1 surface puncta were not static but became enlarged after a long-term depression paradigm. These results support that the synaptic cleft is organized on a nanoscale into sub-compartments marked by distinct trans-synaptic complexes.

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 1-3nm. 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 (∼400nm) 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-ß-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.

Robust membrane detection based on tensor voting for electron tomography.

Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure.