Biology subjects

Steyer, A. M.

Publications and source records attributed to Steyer, A. M..

15 recordsLinked to original sources

bioRxiv2026

Intracellular genomic variability driven by cellular compartmentalization in the giant bacterium Achromatium spp

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 cells 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.

microbiology↗

bioRxiv2026

An Advanced Mobile Laboratory to enable field-based microbial ecology and cell biology across scales

Microbial biodiversity is central to ecosystem function, yet mechanistic insights into the cell biology of environmental organisms remain limited. The underlying challenges are twofold: most microbes remain uncultivable, and a persistent gap exists between field sampling and laboratory analyses. Here, we introduce the Advanced Mobile Laboratory (AML), a field-deployable platform that integrates confocal microscopy, image-enabled cell sorting, and cryo-preparation for expansion and electron microscopy. This setup enables immediate, standardized processing and analysis of environmental communities directly at the sampling site. We demonstrate its capability using marine eukaryotic plankton, showing how the AML enables multiscale investigations, from live imaging of natural communities to enabling ultrastructural and single-cell omics analyses, while minimizing sample degradation and enabling on-site experimentation. By bringing high-end sample preparation and analytical capacity into the field, the AML enables studying life in its natural context to mechanistically understand lifes diversity in the environment.

cell biology↗

bioRxiv2025

The In Situ Structure and Distribution of V-ATPase in C. elegans Apical Membrane Stacks

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.

molecular biology↗

bioRxiv2025

Tether-mediated extraction of myelinoid bodies by microglia and astrocytes can maintain myelin integrity

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.

neuroscience↗

bioRxiv2025

Organisation of axial regions of isolated mitotic chromosomes visualised by cryo correlative light and electron tomography

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.

cell biology↗

bioRxiv2025

Developmental axon diameter growth of central nervous system axons does not depend on ensheathment or myelination by oligodendrocytes

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 (Mbpshi/shi and Myrf 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.

neuroscience↗

bioRxiv2023

Targeted volume Correlative Light and Electron Microscopy of an environmental marine microorganism

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods, along with the complexity and heterogeneity of environmental samples, pose great technical challenges. In the present study, we used a workflow based on a specific EM sample preparation, compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate with quantitative characterization of multiple organelles. We could show that this cell contains a single convoluted chloroplast and the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed. Together with providing insights in dinoflagellates biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

cell biology↗

bioRxiv2022

Age-dependent structural reorganization of utricular ribbon synapses

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, a direct link to the functional decline of VHC ribbon synapses remains to be demonstrated. To address this issue, we investigated the effects of aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to one year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

cell biology↗

bioRxiv2022

Piccolino regulates the architecture of the ribbon at cochlear inner hair cell synapses

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tireless-ly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for precise sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterized the structure and function of IHC-synapses in piccolo gene-trap mutant rats (Pclogt/gt). We found a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology were altered in apical IHCs, while their presynaptic function seemed unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal synaptic transmission.

neuroscience↗

bioRxiv2021

Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here we find that expression of the tetraspan-transmembrane protein CMTM5 (Chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and CNS myelin. Genetic disruption of the Cmtm5-gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5-deficiency causes an early-onset progressive axonopathy, which we also observe in global and in tamoxifen-induced oligodendroglial Cmtm5-mutants. Presence of the Wlds mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

neuroscience↗

bioRxiv2021

Structural analysis of the C. elegans dauer larval anterior sensilla by Focused Ion Beam-Scanning Electron Microscopy

At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include: 1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; 2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; 3) variability in amphid sensory dendrites among dauers; 4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.

neuroscience↗

bioRxiv2021

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

Correlative light and electron microscopy (CLEM) combines two imaging modalities, balancing out the limits of one technique with the other. In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume acquisition at the ultrastructural level. We present a toolset for adherent cultured cells that enables tracking and finding cell regions previously identified in light microscopy, in the FIB-SEM along with automatic acquisition of high-resolution volume datasets. We detect a grid pattern in both modalities (LM and EM), which identifies common reference points. The novel combination of these techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscopes field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 {micro}m accuracy, while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible.

cell biology↗

bioRxiv2020

White matter integrity requires continuous myelin synthesis at the inner tongue

Myelin, the electrically insulating axonal sheath, is composed of lipids and proteins with exceptionally long lifetime. This raises the question how myelin function is affected by myelin turnover. We have studied the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survived recombination, continued expressing myelin genes, but failed to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualized myelin-like membranes that failed to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicated degradation of existing myelin at the abaxonal side and at the inner tongue of the sheath. Compacted myelin thinning and shortening of internodes, with about 50% myelin lost after 20 weeks (=5 months), ultimately led to axonal pathology and neurological disease. These data reveal that functional axon-myelin units require the continuous incorporation of new myelin membranes.

neuroscience↗

bioRxiv2019

Macromolecular and electrical coupling between inner hair cells in the rodent cochlea

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, 30% of the IHCs are electrochemically coupled in mini-syncytia. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D-electron-microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

neuroscience↗

bioRxiv2017

Postmitotic Nuclear Pore Assembly Proceeds By Radial Dilation Of Small ER Membrane Openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

cell biology↗