Vojtěch Pražák

Genome function in the nucleus is organised through membrane-less compartments enriched with specific proteins and nucleic acids. Promyelocytic leukemia (PML) bodies regulate telomere maintenance and DNA damage responses, but their internal molecular organisation remains poorly understood. We visualised the molecular composition of PML bodies directly within the nucleus using an advanced cryogenic-correlative light and electron microscopy (cryo-CLEM) pipeline. Cryo-electron tomography revealed eYFP-PML-I bodies as compartments with a molecular makeup distinct from the surrounding nucleoplasm. Cryogenic super-resolution correlative light and electron microscopy showed these comprise a diffuse PML-protein shell enclosing an inner core. A visual proteomics comparison of the core and surrounding regions through template matching and sub-tomogram averaging mapped nucleosomes, TRiC chaperonin complexes in both closed and open conformations, and single-capped PA28 proteasomes. Membrane structures of unknown function were additionally detected within some eYFP-PML-I bodies. Remarkably, the nucleosomes included density consistent with columnar trinucleosome assemblies (1), revealing that PML body interiors harbour discrete chromatin domains. Moreover, vault complexes were detected in poised positions in the proximal nucleoplasm. Together, these first in situ higher-resolution structural insights of PML bodies identify their critical functional components and an interior porous architecture that suggests a mechanism for content selection based on physical partitioning akin to size-exclusion chromatography.

Electron cryo-tomography (cryoET) resolves cellular structure in three dimensions, yet region selection is still typically based on two-dimensional projection images. Here, we introduce View Tomo, a workflow for rapid acquisition of low-magnification tomograms that enables screening, targeting and analysis in 3D. View Tomo tilt series are acquired in minutes at low dose (~3 e ⁻ /Å ² ), producing high-contrast tomograms that remain compatible with subsequent high-resolution structural determination. We implemented View Tomo using an automated acquisition and reconstruction pipeline for rapid alignment. Across multiple viral and cellular systems, view tomograms revealed membrane remodelling events, assembly intermediates and cellular organisation that are difficult to identify in projection images. These data enabled targeted high-resolution imaging and quantitative analysis of spatial relationships within cells. View Tomo therefore extends cryoET workflows by improving target selection, enabling analysis of mesoscale organisation, and facilitating integration with correlative imaging approaches.

Cryogenic correlative light and electron microscopy (cryo-CLEM) combines specific fluorescence labelling of proteins inside cells with structural information at the angstrom-level. The introduction of super-resolution fluorescence methods in the field of cryogenic fluorescence microscopy is a necessary step to bridge the large resolution gap between the different imaging modalities. However, there are many challenges hindering the full potential of cryogenic super-resolution correlative light and electron microscopy and seamless integration with structural cell biology. One of the main limiting factors is a lack of dedicated cryogenic fluorescence microscopy systems with sufficient mechanical stability to enable the collection of high-quality super-resolution data and full compatibility with vitrified specimens for cryo-electron tomography. Here, we address this by developing a vacuum-free ultra-stable cryogenic optical microscope (VULCROM). VULCROM is a dedicated super-resolution cryo-CLEM (cryo-SR-CLEM) setup that combines the stability of a vacuum-insulated cryostat with the flexibility and modularity of an open microscopy system. We demonstrate that VULCROM enables detailed investigations of single-molecule cryo-photo-physics across timescales spanning milliseconds to hours. We furthermore demonstrate its suitability for routine cryo-SR-CLEM with a resolution in the 10 nm range in distinct vitrified biological specimen types. We resolve the nanoscale architecture of YFP-labelled PML bodies within the nucleus of mammalian cells and the distribution of ATG9-eGFP in its cellular structural context in a cryo-lift-out lamella of N. benthamiana plant tissue. Owing to its vacuum-free design, VULCROM can be readily adapted for diverse correlative workflows and other cryo-light microscopy applications.

Communication between cellular organelles is essential for mounting effective innate immune responses. The transport of organelles to pathogen penetration sites and their assembly around the host membrane, which delineates the plant-pathogen interface, are well-documented. However, whether organelles associate with these specialized interfaces, and the extent to which this process contributes to immunity, remain unknown. Here, we discovered defense-related membrane contact sites (MCS) comprising a membrane tethering complex between chloroplasts and the extrahaustorial membrane (EHM) surrounding the haustorium of the pathogen Phytophthora infestans in Nicotiana benthamiana. The assembly of this complex involves association between the chloroplast outer envelope protein CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) and its plasma membrane-associated partner KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1). Our biochemical assays revealed that CHUP1 and KAC1 interact, and infection cell biology assays demonstrated their co-accumulation in foci where chloroplasts contact the EHM. Genetic depletion of CHUP1 or KAC1 reduces the focal deposition of callose around the haustorium without affecting other core immune processes. Our findings suggest that the chloroplast-EHM attachment complex promotes plant focal immunity, revealing key components and their potential roles in the deposition of defense materials at the pathogen interface. These results advance our understanding of organelle-mediated immunity and highlight the significance of MCS in plant-pathogen interactions.

Summary paragraph: Viruses are obligate intracellular parasites and viral infections lead to massive host cell rearrangement to support the rapid generation of progeny. Host take-over and remodelling include formation of viral-induced compartments for viral genome replication and/or assembly. While viruses infecting bacteria, bacteriophages or phages, have been extensively characterized in vitro , the molecular mechanisms underlying the viral cycle inside the crowded cytoplasm remain unclear. Here, we investigate the spatial reorganization of SPP1-infected bacteria under near-native conditions by electron cryo tomography. The most prominent feature is the formation of a large viral DNA (vDNA) compartment from which ribosomes are excluded. In SPP1 infection, there is no membrane nor proteinaceous shell surrounding these compartments. Also, we identified novel key intermediates in virus assembly: open precursors of procapsid lattice were found at the cytoplasmic membrane in a process that requires expression of the portal protein. Next, DNA-free procapsids relocate inside the vDNA compartment where vDNA is packed in a stepwise manner. Finally, DNA-filled capsids segregate to the periphery of the compartment for assembly completion and storage. Collectively, we provide comprehensive mechanistic insights into the complete viral assembly pathway of SPP1 directly in cellula and show how specific steps are coordinated inside the reorganized bacterial cell.

The dynamic regulation of mitochondria shape via fission and fusion is critical for cellular responses to stimuli. In homeostatic cells, two modes of mitochondrial fission, midzone and peripheral, provide a decision fork between either proliferation or clearance of mitochondria. However, the relationship between specific mitochondria shapes and functions remains unclear in many biological contexts. While commonly associated with decreased bioenergetics, fragmented mitochondria paradoxically exhibit elevated respiration in several disease states, including infection with the prevalent pathogen human cytomegalovirus (HCMV) and metastatic melanoma. Here, incorporating super-resolution microscopy with mass spectrometry and metabolic assays, we use HCMV infection to establish a molecular mechanism for maintaining respiration within a fragmented mitochondria population. We establish that HCMV induces fragmentation through peripheral mitochondrial fission coupled with suppression of mitochondria fusion. Unlike uninfected cells, the progeny of peripheral fission enter mitochondria-ER encapsulations (MENCs) where they are protected from degradation and bioenergetically stabilized during infection. MENCs also stabilize pro-viral inter-mitochondria contacts (IMCs), which electrochemically link mitochondria and promote respiration. Demonstrating a broader relevance, we show that the fragmented mitochondria within metastatic melanoma cells also form MENCs. Our findings establish a mechanism where mitochondria fragmentation can promote increased respiration, a feature relevant in the context of human diseases.

Actin-based motility is required for the transmission of malaria sporozoites. While this has been shown biochemically, filamentous actin has remained elusive and has to date never been directly visualised inside the parasite. Using focused ion beam milling and electron cryo-tomography, we studied dynamic actin filaments in unperturbed Plasmodium falciparum cells for the first time. This allowed us to dissect the assembly, path and fate of actin filaments during parasite gliding and determine a complete 3D model of F-actin within sporozoites. We show that within the cell, actin assembles into micrometre long filaments, much longer than observed in in vitro studies. After their assembly at the parasite’s apical end, actin filaments continue to grow as they are transported down the cell as part of the glideosome machinery, and are disassembled at the basal end in a rate-limiting step. Large pores in the IMC, constrained to the basal end, may facilitate actin exchange between the pellicular space and the cytosol for its recycling and maintenance of directional actin flow for efficient gliding. The data also reveal striking and extensive actin bundles in the nucleus. Implications of these structures for motility and transmission are discussed.

Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV.

The nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without breaking the nuclear envelope barrier. It assembles into a lattice on the inner nuclear membrane enveloping newly assembled nucleocapsids, which bud into the perinuclear space. The primary virion envelope subsequently fuses with the outer nuclear membrane, releasing the capsid into the cytosol. Here we interrogated the NEC in the context of intact cells infected with pseudorabies or herpes simplex virus using focused-ion beam milling and electron cryo- tomography. We determined the structure of NEC in different conformations and show that it consists of a flexible hexameric lattice that generates curvature through a combination of ordered and disordered domains. After interrogating the intermediate stages of capsid formation, we show that capsid vertex binding may initiate envelopment but does not directly induce curvature formation. These data and many examples of the intermediate stages of nuclear egress paint a detailed holistic view of a versatile transport system.

Although vaccinia virus (VACV) is the best studied poxvirus, the structure of the mature virus (MV) remains poorly understood. Its asymmetric shape, size and compactness poses a major challenge for electron microscopy (EM) analysis, including cryoEM. Sub-viral particles, in particular membrane-free viral cores, may overcome these limitations. We compare cores obtained by detergent-stripping MVs with cores in the cellular cytoplasm, early in infection. By combining cryo-electron tomography (cryoET), subtomogram averaging (STA) and AlphaFold2 (AF2), abundant core-structures are analyzed, focusing on the prominent palisade layer on the core surface. On detergent-stripped cores, the palisade is composed of densely packed trimers of the major core protein A10. On the core surface they display a random order and their classification indicate structural flexibility. On cytoplasmic cores A10 is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. CryoET and STA also uncover unexpected details of the layers beneath the palisade both on in vitro and in situ cores, that are compared to AF2 structure predictions of known VACV core-associated proteins. Altogether, our data identify for the first time the structure and molecular composition of the palisade units. The results are discussed in the context of the VACV replicative cycle, the assembly and disassembly of the infectious MV.