Biology subjects

Falckenhayn, J.

Publications and source records attributed to Falckenhayn, J..

3 recordsLinked to original sources

bioRxiv2026

In situ molecular architecture of PML bodies reveals open-state columnar trinucleosome assemblies within a porous, chromatin-permissive interior

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/729032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19ad697org.highwire.dtl.DTLVardef@7e8590org.highwire.dtl.DTLVardef@72f0caorg.highwire.dtl.DTLVardef@1e8a1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

View Tomo: Context-aware targeting and analysis in electron cryo-tomography

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-/[A]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.

cell biology↗

bioRxiv2026

On-lamella super-resolution cryo-CLEM for cryo-ET enabled by vacuum-free ultra-stable cryogenic fluorescence microscopy

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.

biophysics↗