Anna Maria Steyer

Bacteria of the genus Achromatium harbor hundreds of chromosomes that were previously suggested to be genetically diverse. By sequencing multiple regions from individual cells, we demonstrate that chromosomes within a single cell differ in nucleotide and amino acid sequence, reaching levels of divergence well below accepted bacterial species boundaries and below the range associated with homologous recombination. Sequencing of dividing cells further revealed that daughter cells inherit distinct chromosome populations, a mode of inheritance previously associated with sexual reproduction in eukaryotes. Combining subcellular sequencing with high-resolution microscopy, we reconstruct the three-dimensional cellular architecture of Achromatium and show that extensive intracellular heterogeneity arises from the spatial segregation of chromosome populations by the cell’s internal structure, which limits genome-wide recombination. Our findings establish cellular architecture as a determinant of genome evolution in giant polyploid bacteria and identify spatial genome segregation as a mechanism enabling the maintenance and inheritance of divergent chromosome populations in bacteria.

Biological imaging data are a valuable resource for life science research, but their long-term value depends on effective data management. This tutorial introduces key concepts and best practices for managing imaging data throughout the research lifecycle, including standards such as OME-Zarr, public repositories, REMBI, and ontologies. Participants will explore the principles of FAIR data and discover how community-driven approaches can improve the findability, accessibility, interoperability, and reuse of imaging data.

Cryo-correlative light and electron microscopy methods enable targeted structural analysis of fluorescently labelled features in vitrified specimens. However, correlative workflows on high-pressure frozen samples often remain challenging due to the lack of persistent landmarks for reliable sample tracking and image registration between different microscopes. Standard high-pressure freezing carriers provide little intrinsic reference information, as the exposed sample surface is often smooth and rotationally ambiguous, complicating localisation of regions of interest across imaging platforms. Here, we introduce PinCorr, a 3-mm high-pressure freezing carrier with an integrated coordinate system formed by four asymmetrically arranged pillars with distinct geometries. These built-in landmarks remain visible after freezing and provide a stable, sample-independent reference frame for orientation and correlation between cryo-fluorescence microscopy and electron microscopy. We show that PinCorr supports fluorescence-guided cryo-volume imaging, serial lift-out for cryo-electron tomography and freeze-substitution workflows followed by room-temperature on-section correlation. PinCorr thus provides a hardware-based approach to establishing a persistent spatial reference frame in HPF-based correlative imaging workflows for thick and multicellular specimens.

Dysregulated intracellular lipid metabolism emerged as a driver of heart failure (HF), a leading cause of death worldwide that is frequently caused by dilated cardiomyopathy (DCM). Yet, how defective lipid signaling destabilizes failing cardiomyocytes (CMs) at the molecular level remains elusive. We utilized induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), patient-derived heart tissue, and living adult CMs to elucidate lipid-dependent endoplasmic reticulum (ER) dysregulation in DCM and HF and identify new therapeutic targets. Lipidomics revealed abnormal intracellular cholesterol in iPSC-CMs carrying DCM-causing mutations in sarcomere proteins (tropomyosin; troponin T). Elevated cholesterol in CMs was found linked to abnormal ER architecture and dysfunction. STED microscopy, electron tomography, and biochemical analyses demonstrated that pathological ER remodeling and abnormal curvature are triggered by loss of ER-sarcomere/cytoskeleton contacts due to sarcomere misalignment in DCM CMs. Mechanistically, this signaling axis levers ER membrane dysfunctions and abnormal cholesterol levels via ER-sarcomere contact sites and operates in a bidirectional manner. Restoring intracellular cholesterol balance rescued ER membrane remodeling, sarcomere-ER contact site signaling, and sarcomere disarray in DCM iPSC-CMs. This ultimately improved the defective contractility of DCM iPSC-CMs, a key feature of failing CMs that contain a misaligned, dysfunctional sarcomere cytoskeleton. We validated this pathomechanism in end-stage DCM hearts and living atrial cardiomyocytes. Our findings suggest that intracellular cholesterol functions as a conserved ER membrane modulator and structural determinant across human CMs. Taken together, we present the lipidomic landscape of DCM and identify defective ER/cholesterol signaling as a disease driver whose therapeutic targeting rescues key functions in failing CMs.

The cuticle of Caenorhabditis elegans is an apical extracellular matrix composed primarily of collagens which protects the organism from external stresses and facilitates mobility. The cuticle directly contacts several epithelial cell structures including regular, repeating folds of the apical plasma membrane, known as apical membrane stacks (AMSs). Here, we examine the structure of AMSs using in situ cryo-electron tomography (cryo-ET). We find that V-ATPases are distributed across the cytoplasmic-facing membranes of the AMSs. We then determine the in situ structure of V-ATPase and show that it is highly conserved relative to other eukaryotic V-ATPases. Analysis of the distribution of V-ATPases on the AMSs shows that they form clusters in which individual V-ATPases are tightly packed but do not oligomerise. Our data also show that complete, fully assembled V-ATPases line the AMSs. Previous studies have established that V-ATPases can only act as ATP-dependent proton pumps in their fully assembled state. Our findings indicate that V-ATPases arranged along the AMSs pump protons directly into the extracellular matrix, suggesting that a primary function of AMSs is to acidify the cuticle.

Oligodendrocytes make myelin for the electrical insulation of axons and saltatory impulse conduction. Myelin lipids and proteins undergo a slow turnover, but exactly how the multilamellar and compacted membrane sheaths are remodeled without compromising myelin sheath integrity has remained puzzling, in particular at advanced age when myelin abnormalities increase. Earlier EM studies had suggested myelin membranes are shed and subsequently phagocytosed by microglia. However, the formation of multilamellar myelinoid bodies (MBs), leaving a well-ordered myelin sheath behind, is difficult to reconcile with simple shedding mechanisms. Here, we show by three-dimensional FIB-SEM reconstructions of optic nerves in mice and by two-photon live-imaging of myelinated cortical slices that MBs are initially connected to their parental sheaths by long tethers, which are stretched by trogocytosing microglia and astrocytes. We observe ruptured tethers attached to both MBs and sheaths, suggesting a novel mechanism of tension-driven tether scission. Importantly, the successive fusion of the corresponding innermost myelin membranes in an extended tether can preserve myelin sheath integrity. Thus, the remodeling by tether-mediated MB extraction emerges as a mechanism of physiological maintenance of myelin sheaths in the CNS.

BioStudies – one package for all the data supporting a study

Understanding the intracellular fate of nanoparticles (NPs) is essential for advancing nanomedicine, particularly in targeted drug delivery for cancer therapy. Here, we present a complementary cryogenic microscopy workflow across scales to investigate the uptake and subcellular localization of zirconyl‐containing inorganic–organic hybrid nanoparticles (IOH‐NPs) in murine breast cancer cells. Our approach integrates cryogenic fluorescence microscopy (cryo‐FM), cryo‐focused ion beam scanning electron microscopy (cryo‐FIBSEM), and cryo‐soft X‐ray tomography (cryo‐SXT), enabling molecular specificity, high‐resolution imaging, and volumetric ultrastructural analysis in near‐native cellular states. We demonstrate that the cryogenic workflow provides enough contrast and resolution across all modalities for quantifying the IOH‐NP uptake: NPs are internalized within 2 h of incubation and progressively accumulate in endolysosomes over time, as confirmed by fluorescence labeling and SXT. Quantitative analysis reveals a marked increase in endolysosomal accumulation of IOH‐NPs from 2 to 24 h. Our findings help to establish multimodal cryogenic microscopy as a powerful tool for nanoscale imaging and quantitative analysis of NP uptake within close‐to‐native cells, offering new insights into NP trafficking and cellular responses relevant to nanomedicine development.

The formation of mitotic chromosomes is essential for the accurate segregation of genetic material during cell division. Increasing evidence suggests that chromosome formation involves the reorganization of DNA into loops anchored within chromosomal axial regions, whose structural organization remains insufficiently characterized. Taking advantage of DT40 cells, an avian cell model characterized by the presence of a range of chromosome sizes from 3.2-197 Mb, we have established a preparation of entire close-to-native native mitotic chromosomes for cryogenic correlative light and electron microscopy (cryo-CLEM). The size of the smallest chromosomes allows imaging of their axial regions without further thinning. Cryo-electron tomography of the chromosome axial regions reveals the presence of heterogeneous non-histone macromolecular densities (NHMDs), approximately 30–45 nm in size, interspersed within chromatin/DNA regions. We propose that NHMDs may contain condensins and contribute to chromosome architecture. In addition to NHMDs, we identified dense clusters of particles, similar in size, near the chromosome surface, likely associated with ribosomal components. To quantitatively differentiate NHMDs from these surface clusters, we developed an analytical approach based on particle interspacing and spatial distribution within the chromosome volume. By establishing a cryo-CLEM workflow for whole, near-native mitotic chromosomes, our study provides a foundation for investigating their ultrastructural architecture.

SUMMARY: Myelination facilitates the rapid conduction of action potentials along axons. In the central nervous system (CNS), myelinated axons vary over 100-fold in diameter, with conduction speed scaling linearly with increasing diameter. Axon diameter and myelination are closely interlinked, with axon diameter exerting a strong influence on myelination. Conversely, myelinating Schwann cells in the peripheral nervous system can both positively and negatively affect axon diameter. However, whether axon diameter is regulated by CNS oligodendrocytes is less clear. Here, we investigated CNS axon diameter growth in the absence of myelin using mouse ( Mbp ^shi/shi and M yrf conditional knockout) and zebrafish ( olig2 morpholino) models. We find that neither the ensheathment of axons, nor the formation of compact myelin are required for CNS axons to achieve appropriate and diverse diameters. This indicates that developmental CNS axon diameter growth is independent of myelination, and shows that myelinating cells of CNS and PNS differentially influence axonal morphology.