Scalable electron tomography for connectomics.

We demonstrate limited-tilt, serial section electron tomography (ET), which can non-destructively map brain circuits over large 3D volumes and reveal high-resolution, supramolecular details within subvolumes of interest. We show accelerated ET imaging of thick sections (>500 nm) with the capacity to resolve key features of neuronal circuits including chemical synapses, endocytic structures, and gap junctions. Furthermore, we systematically assessed how imaging parameters affect image quality and speed to enable connectomic-scale projects.

New rabies viral resources for multi-scale neural circuit mapping.

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

Cristae formation is a mechanical buckling event controlled by the inner mitochondrial membrane lipidome.

Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy.

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories.

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis.

Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss of function causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome.

Cristae are high curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous mechanisms for lipids have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the IMM against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. The model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that CL is essential in low oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of CL is dependent on the surrounding lipid and protein components of the IMM.

A Cristae-Like Microcompartment in Desulfobacterota.

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species.

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.

A Uniform and Isotropic Cytoskeletal Tiling Fills Dendritic Spines.

Dendritic spines are submicron, subcellular compartments whose shape is defined by actin filaments and associated proteins. Accurately mapping the cytoskeleton is a challenge, given the small size of its components. It remains unclear whether the actin-associated structures analyzed in dendritic spines of neurons in vitro apply to dendritic spines of intact, mature neurons in situ. Here, we combined advanced preparative methods with multitilt serial section electron microscopy (EM) tomography and computational analysis to reveal the full three-dimensional (3D) internal architecture of spines in the intact brains of male mice at nanometer resolution. We compared hippocampal (CA1) pyramidal cells and cerebellar Purkinje cells in terms of the length distribution and connectivity of filaments, their branching-angles and absolute orientations, and the elementary loops formed by the network. Despite differences in shape and size across spines and between spine heads and necks, the internal organization was remarkably similar in both neuron types and largely homogeneous throughout the spine volume. In the tortuous mesh of highly branched and interconnected filaments, branches exhibited no preferred orientation except in the immediate vicinity of the cell membrane. We found that new filaments preferentially split off from the convex side of a bending filament, consistent with the behavior of Arp2/3-mediated branching of actin under mechanical deformation. Based on the quantitative analysis, the spine cytoskeleton is likely subject to considerable mechanical force in situ.

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy.

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization.

The Parkinson's-disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that regulators of the LRRK2 signaling pathway including vps35 and PPM1H converge upon causing centrosomal deficits. The cohesion alterations do not require the presence of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43 or the presence of RILPL2. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendage of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab proteins to cause the centrosomal defects. The centrosomal alterations impair cell polarization as monitored by scratch wound assays which is reverted by LRRK2 kinase inhibition. These data reveal a common molecular pathway by which enhanced LRRK2 kinase activity impacts upon centrosome-related events to alter the normal biology of a cell.

Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2.

NFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin ß1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

Morphological principles of neuronal mitochondria.

In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like adenosine triphosphate and heat often represent mitochondria as idealized geometries, and therefore, can miscalculate the metabolic fluxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial transmission electron microscopy (TEM) tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.64 nanometer isotropic voxels. The resulting in-silico representations were subsequently quantified by differential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs present across this population of mitochondria, which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons.

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses.

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.

Public mobility data enables COVID-19 forecasting and management at local and global scales.

Policymakers everywhere are working to determine the set of restrictions that will effectively contain the spread of COVID-19 without excessively stifling economic activity. We show that publicly available data on human mobility-collected by Google, Facebook, and other providers-can be used to evaluate the effectiveness of non-pharmaceutical interventions (NPIs) and forecast the spread of COVID-19. This approach uses simple and transparent statistical models to estimate the effect of NPIs on mobility, and basic machine learning methods to generate 10-day forecasts of COVID-19 cases. An advantage of the approach is that it involves minimal assumptions about disease dynamics, and requires only publicly-available data. We evaluate this approach using local and regional data from China, France, Italy, South Korea, and the United States, as well as national data from 80 countries around the world. We find that NPIs are associated with significant reductions in human mobility, and that changes in mobility can be used to forecast COVID-19 infections.