Integrating cellular electron microscopy with multimodal data to explore biology across space and time.

Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces of this spectrum but can be combined to construct a more holistic view. In this Review, we detail the latest advancements in volume electron microscopy (vEM) and cryo-electron tomography (cryo-ET), which together can visualize biological complexity across scales from the organization of cells in large tissues to the molecular details inside native cellular environments. In addition, we discuss emerging methodologies for integrating three-dimensional electron microscopy (3DEM) imaging with multimodal data, including fluorescence microscopy, mass spectrometry, single-particle analysis, and AI-based structure prediction. This multifaceted approach fills gaps in the biological continuum, providing functional context, spatial organization, molecular identity, and native interactions. We conclude with a perspective on incorporating diverse data into computational simulations that further bridge and extend length scales while integrating the dimension of time.

Piecing together the structural organisation of lipid exchange at membrane contact sites.

Membrane contact sites (MCSs) are areas of close proximity between organelles, implicated in transport of small molecules and in organelle biogenesis. Lipid transfer proteins at MCSs facilitate the distribution of lipid species between organelle membranes. Such exchange processes rely on the apposition of two different membranes delimiting distinct compartments and a cytosolic intermembrane space. Maintaining organelle identity while transferring molecules therefore implies control over MCS architecture both on the ultrastructural and molecular levels. Factors including intermembrane distance, density of resident proteins, and contact surface area fine-tune MCS function. Furthermore, the structural arrangement of lipid transfer proteins and associated proteins underpins the molecular mechanisms of lipid fluxes at MCSs. Thus, the architecture of MCSs emerges as an essential aspect of their function.

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

The architecture of Cidec-mediated interfaces between lipid droplets.

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 µm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs.

Ultrastructure of COPII vesicle formation in yeast characterized by correlative light and electron microscopy.

Traffic of proteins out of the endoplasmic reticulum (ER) is driven by the COPII coat, a layered protein scaffold that mediates the capture of cargo proteins and the remodeling of the ER membrane into spherical vesicular carriers. Although the components of this machinery have been genetically defined, and the mechanisms of coat assembly extensively explored in vitro, understanding the physical mechanisms of membrane remodeling in cells remains a challenge. Here we use correlative light and electron microscopy (CLEM) to visualize the nanoscale ultrastructure of membrane remodeling at ER exit sites (ERES) in yeast cells. Using various COPII mutants, we have determined the broad contribution that each layer of the coat makes to membrane remodeling. Our data suggest that inner coat components define the radius of curvature, whereas outer coat components facilitate membrane fission. The organization of the coat in conjunction with membrane biophysical properties determines the ultrastructure of vesicles and thus the efficiency of protein transport.

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion.

Cell-cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h- isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h- cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h- cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h- cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

Closed mitosis requires local disassembly of the nuclear envelope.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles.

Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2-4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7-9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.

Cargo crowding contributes to sorting stringency in COPII vesicles.

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.

Membrane Architecture in the Spotlight of Correlative Microscopy.

Cellular membranes differ in their molecular organisation, shape, and dynamics. Knowing how these properties of membrane architecture relate to the presence and function of specific membrane components is fundamental for understanding membrane-associated cellular processes. Correlative light and electron microscopy (CLEM) is ideally poised to address such problems. Fluorescence microscopy allows identification of cellular membranes through labelled components and can provide temporal information, while electron microscopy allows visualisation of the structure of the same membranes at high resolution. In recent years, various CLEM protocols have been applied to gain insights into cellular membrane architecture. Here, we review conceptually novel approaches by which CLEM has provided insights on membrane reshaping, subcellular localisation of components, host-pathogen interactions, and has answered longstanding mechanistic questions.

Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures.

Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways.

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis.

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation.

Perspective on architecture and assembly of membrane contact sites.

Membrane contact sites (MCS) are platforms of physical contact between different organelles. They are formed through interactions involving lipids and proteins, and function in processes such as calcium and lipid exchange, metabolism and organelle biogenesis. In this article, we discuss emerging questions regarding the architecture, organisation and assembly of MCS, such as: What is the contribution of different components to the interaction between organelles? How is the specific composition of different types of membrane contacts sites established and maintained? How are proteins and lipids spatially organised at MCS and how does that influence their function? How dynamic are MCS on the molecular and ultrastructural level? We highlight current state of research and point out experimental approaches that promise to contribute to a spatiomechanistic understanding of MCS functions.

Meeting report - Emerging Concepts in Cell Organization.

New concepts in cell organization emerged in a medieval castle during a snowy week in January 2017 in the middle of the Austrian Alps. The occasion was the 10th Annaberg EMBO workshop in Goldegg am See; organized by Gabriele Seethaler, Catherine Rabouille and Marino Zerial. There were 95 participants, including many who gave talks and presented posters, enjoying a familial and trusting atmosphere that stimulated lively exchange of (unpublished) results, new ideas and thoughts.