Biology subjects

Helmstaedter, M.

Publications and source records attributed to Helmstaedter, M..

17 recordsLinked to original sources

bioRxiv2026

M-CSF drives alveolar macrophage plasticity during development and cytomegalovirus infection

Alveolar macrophages (AM), the most frequent resident immune cells of the lung, are at the first line of defence against respiratory pathogens and instruct structural lung cells, e.g. in tissue repair. They are long-lived and receive their terminal phenotypic imprint through signals originating from the unique location at the tissue-air interface, as well as through cytokines like granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-{beta} (TGF-{beta}). However, the regulatory mechanisms governing their phenotypic plasticity, which is conceptually critical for their positioning and differentiation in early life and for their functional adaptation during infection, remain poorly defined. Here we explored respiratory tract infection with cytomegalovirus (CMV), which is closely linked to mammalian immune evolution. Complementary host-pathogen fate-mapping strategies revealed AM to constitute the bottleneck for efficient mouse (M)CMV infection. MCMV infection induced macrophage colony-stimulating factor (M-CSF) in the alveolar space, and culturing of AM in M-CSF led to a profound remodelling of morphology, immunophenotype, and transcriptional identity, e.g. it increased the expression of interferon-stimulated genes (ISG), which modulated susceptibility to infection. Notably, already at baseline recently differentiated neonatal AM across species retained an M-CSF-associated transcriptional program. This was linked to reduced permissiveness to respiratory MCMV infection in vivo. Overall, our findings identify the role of M-CSF-dependent signalling in conferring plasticity to AM, when it is most needed, particularly during early-life establishment and in response to viral infection.

immunology↗

bioRxiv2026

Fibroblast-derived Collagen VI shapes the structure and function of the tumor-immune microenvironment in clear cell renal cell carcinoma

The importance of the extracellular matrix (ECM) influencing tumor biology in stroma-rich tumors is well established. However, the relevance of individual ECM proteins in rather stroma-poor cancers such as clear cell renal cell carcinoma (ccRCC) is ill-defined. Using bulk proteomics, spatial imaging, and single-cell transcriptomics, we identify collagen VI (COL6) as a predominant ECM component of the ccRCC interstitial stroma, synthesized primarily by fibroblasts and pericytes. Using cell-derived matrix (CDM) models, we demonstrate that COL6 is essential for maintaining an isotropic ECM network architecture and governs the broader matrisomal composition, with direct pro-proliferative consequences for tumor cells both in vitro and in situ. Granular spatial analysis reveals that COL6-rich stromal septa constrain tumor-infiltrating T cells to boundary zones, where CD8+PD1+ phenotypes predominate. Importantly, tyrosine kinase inhibition (TKI) with cabozantinib suppresses COL6 expression in fibroblasts in vitro and in ex vivo tumor models, mirroring COL6-depleted CDM phenotypes. Our findings establish COL6 as a central stromal regulator of ccRCC tumor biology and immune contexture, revealing ECM remodeling as an underappreciated mechanism of TKI action, with implications for combination immunotherapy strategies.

cancer biology↗

bioRxiv2025

The Second Mitochondrial Activator of Caspases (SMAC) regulates growth, inflammation and mitochondrial integrity in cancer cells

SMAC is a mitochondrial intermembrane space protein, which is released during apoptosis and whose known function is antagonism of inhibitor of apoptosis proteins in the cytosol, to facilitate caspase activation. Recent data suggest that SMAC can also be released by sub-lethal signals in the apoptosis pathway, in the absence of cell death. We here explored potential functions of SMAC in non-apoptotic cells. We found that a portion of SMAC is spontaneously released into the cytosol in the absence of apoptosis, regulated by the BCL-2-family proteins BAX and BAK and the fission GTPase DRP1. In cancer cell lines, SMAC was required for the activation of caspases in lethal and non-lethal conditions, while this contribution to caspase-activation was much smaller in non-malignant fibroblast lines. In cells with high levels of cytosolic SMAC, SMAC deficiency reduced in vitro migration, invasion and anchorage-independent growth. SMAC-deficient cells further showed a reduced activity in interferon signalling, associated with reduced cytosolic presence of mitochondrial DNA and activation of the stimulator of interferon genes (STING), and SMAC expression levels correlated with interferon-induced genes in cancer data sets. We further found that SMAC can regulate mitochondrial morphology and integrity. Finally, high gene-expression of SMAC was associated with poor prognosis in patients of several cancer types. These results identify SMAC as a regulator of inflammation and growth behaviour of cancer cells. They further report a mitochondrial function of SMAC and demonstrate a role of SMAC in human cancer biology across several cancer entities.

cell biology↗

bioRxiv2025

The slit diaphragm in Drosophila features a bi-layered, fishnet-like architecture

The kidney filters large volumes of blood plasma, relying on the glomerulus for the filtration. The slit diaphragm, a critical component of the glomerulus, is formed between podocytes by the immunoglobulin domain proteins nephrin and Neph1. The molecular architecture of the slit diaphragm has remained elusive for decades. Using cryo-electron tomography on focused ion beam-milled Drosophila nephrocytes, an invertebrate podocyte model, we show that the slit diaphragm adopts a fishnet-like pattern. Comparison of hundreds of slit diaphragm segments reveals that it is bi-layered and highly periodic. Based on the cryo-electron tomography map, we propose four possible models for the arrangement of the Drosophila nephrin ortholog (Sns), and the Drosophila Neph1 ortholog (Kirre), the main components of the slit diaphragm in Drosophila. In each model, precise and consistent homo- and heterophilic interactions between crossing immunoglobulin domains of Sns and Kirre become apparent, with immediate implications for the stability and the assembly of the slit diaphragm. Cryo-electron tomography shows that sns silencing disrupts this fishnet pattern, linking this directly to Drosophila nephrin. After Rab5 silencing, causing Sns mistrafficking and ectopic SD formation, the fishnet pattern appears ectopically as well. Our findings align with observations applying cryo-electron tomography to podocytes in mice, indicating that the molecular architecture is evolutionarily conserved across animals. This highlights the value of the nephrocyte as a podocyte model and establishes a crucial link between the architecture of the slit diaphragm and its function.

biophysics↗

bioRxiv2025

A Drosophila model for Dent disease reveals impaired ER export of Cubilin as pathogenic mechanism

Mutations in the CLCN5 gene encoding the chloride-hydrogen exchanger ClC-5 cause Dents disease, a genetic disorder of the endolysosomal pathway in the proximal tubules of the kidneys. Many patients also develop glomerular lesions, but the underlying mechanism is unclear. We have established an in vivo model for Dents disease using Drosophila nephrocytes that share similarities with podocytes and proximal tubular cells. Upon depletion of ClC-c, the fly homologue of CLCN5, the endocytic receptor Cubilin was lost from the cortex of nephrocytes, which led to a strong decrease in albumin uptake and slit diaphragm (SD) turnover. Moreover, the actin and microtubular cytoskeleton as well as Rab11-marked recycling endosomes showed a strong cortical accumulation, whereas cholesterol-enriched autophagic compartments emerged in the perinuclear area. Cubilin exhibited a mild mislocalization to cortical early and late endosomal compartments and, in addition, strongly accumulated in the endoplasmic reticulum (ER). This was accompanied by a fragmentation of the ER morphology and an increase in ER exit sites and associated Golgi stacks. These secretory pathway phenotypes were also observed upon silencing of a subunit of the vacuolar H+-ATPase (V-ATPase) suggesting that they depend on acidification. Therefore, we speculate that ClC-c and the V-ATPase together acidify the Golgi to allow proper glycosylation and surface trafficking of Cubilin (or its binding partner Amnionless). Interestingly, ER retention of Cubilin was confirmed in ClC-5 knockout mice, underscoring the relevance of this pathomechanism for Dents disease. Translational statementIn this work, we study the function of the fly ortholog of CLCN5 whose mutations cause Dents disease, a devastating hereditary kidney disease. By demonstrating that the protein uptake receptor Cubilin is retained in the ER upon ClC-c/ClC-5 depletion in flies and mice, we provide an unexpected new disease mechanism for this disease. Future therapeutic strategies may be directed at improving ER export through acidification of the Golgi apparatus.

cell biology↗

bioRxiv2025

Connectomic analysis of astrocyte-synapse interactions in the cerebral cortex

Astrocytes, a main type of glia cells in the cortex, provide metabolic support to neurons, and their possible function as a synaptic partner has given rise to the notion of "tripartite" synapses, suggesting a contribution to neuronal computations. For astrocytes to serve such purposes, the interactions with synapses in neuronal circuits require a level of specificity beyond overall synaptic support. A systematic mapping of the astrocyte-connectome relationship would enable the testing of these hypotheses - such analysis is however still lacking, in particular for circuits in the cerebral cortex. Here, utilizing previously published connectomic data of more than 200,000 synapses, we systematically analyzed the spatial relation between astrocytes and synapses in mouse somatosensory cortex. We developed a quantitative assessment of astrocyte-synapse proximity, finding that only 22.7% of synapses are contacted by astrocytic processes for more than 50% of their synaptic circumference. This non-ubiquitous astrocytic attachment would render astrocyte-synapse specificity plausible. Astrocytic coverage depended strongly on synapse types, with thalamocortical shaft synapses being the most covered by astrocytic processes. We furthermore observed a strong dependence of astrocytic synaptic coverage on synapse size, which was exclusive for excitatory spine synapses. We then investigated the possible relation of astrocytic synaptic coverage to neuronal activity and synaptic plasticity, finding ultrastructural evidence for substantially reduced astrocytic support at synapses consistent with long-term depression, but not for astrocytic coverage dependence on baseline neuronal presynaptic activity. Together, our data demonstrate a high level of specificity of astrocyte-synapse interactions for particular synaptic types. They indicate the potential relevance of astrocytic coverage for synapse stability, in particular for large synapses, suggesting a contribution to long-term maintenance of learned synaptic states. These methods will allow a systematic testing of hypotheses about glial-neuronal interaction in various brain regions, disease models and species including human.

neuroscience↗

bioRxiv2024

Connectomic reconstruction of a cortical column

The cerebral cortex of mammals has long been proposed to comprise unit-modules, so-called cortical columns. The detailed synaptic-level circuitry of such a neuronal network of about 104 neurons is still unknown. Here, using 3-dimensional electron microscopy, AI-based image processing and automated proofreading, we report the connectomic reconstruction of a defined cortical column in mouse barrel cortex. The cortical column appears as a structural feature in the connectome, without need for geometrical or morphological landmarks. We then used the connectome for definition of neuronal cell types in the column, to determine intracolumnar circuit modules, analyze the logic of inhibitory circuits, investigate the circuits for combination of bottom-up and top-down signals in the column and the specificity of bottom-up and top-down cortical input, search for higher-order circuit structure within homogeneous neuronal populations, and estimate the degree and symmetry of Hebbian learning in the various connection types. With this, we provide a first column-level connectomic description of the cerebral cortex, the likely substrate for a synaptic-level mechanistic understanding of sensory-conceptual integration and learning.

neuroscience↗

bioRxiv2022

RoboEM: automated 3D flight tracing for synaptic-resolution connectomics

Mapping neuronal networks from 3-dimensional electron microscopy data still poses substantial reconstruction challenges, in particular for thin axons. Currently available automated image segmentation methods, while substantially progressed, still require human proof reading for many types of connectomic analyses. RoboEM, an AI-based self-steering 3D flight system trained to navigate along neurites using only EM data as input, substantially improves automated state-of-the-art segmentations and replaces human proof reading for more complex connectomic analysis problems, yielding computational annotation cost for cortical connectomes about 400-fold lower than the cost of manual error correction.

neuroscience↗

bioRxiv2022

Connectomic Analysis of Thalamus-driven Disinhibition in Cortical Layer 4

In mammals, sensory signals are transmitted via the thalamus primarily to layer 4 of the primary sensory cortices. While information about average neuronal connectivity in this layer is available, the detailed and higher-order circuit structure is not known. Here, we used 3-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in a layer 4 barrel. We find that thalamic input drives a subset of interneurons with high specificity. These interneurons in turn target spiny stellate and star pyramidal excitatory neurons with subtype specificity. In addition, they create a directed disinhibitory network directly driven by the thalamic input. Together, this circuit can create differential windows of opportunity for activation of the types of excitatory neurons in dependence of strength and timing of thalamic input. With this, we have identified a so-far unknown degree of specialization of the microcircuitry in the main thalamocortical recipient layer of the primary sensory cortex.

neuroscience↗

bioRxiv2022

High-contrast en-bloc staining of mouse whole-brain samples for EM-based connectomics

Connectomes of human cortical gray matter require high-contrast homogeneously stained samples sized at least 2-3 mm on a side, and a whole-mouse brain connectome requires samples sized at least 5-10 mm on a side. Here we report en-bloc staining and embedding protocols for these and other applications, removing a key obstacle for connectomic analyses at mammalian whole-brain level.

neuroscience↗

bioRxiv2022

Selective endocytosis controls slit diaphragm maintenance and dynamics.

The kidneys generate about 180 liters of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.

cell biology↗

bioRxiv2020

A changing signaling environment induces multiciliated cell trans-differentiation during developmental remodeling

Multiciliated cells (MCCs) are extremely highly-differentiated, presenting >100 cilia and basal bodies. We analyzed how MCCs are lost from the airway-like Xenopus embryonic epidermis during developmental tissue remodeling. We found that some MCCs undergo apoptosis, but that the majority trans-differentiate into secretory cells. Trans-differentiation involves loss of ciliary gene expression, cilia retraction and lysosomal degradation. Apoptosis and trans-differentiation are both induced by a changing signaling environment through Notch, Jak/STAT, Thyroid hormone and mTOR signaling, and trans-differentiation can be inhibited by Rapamycin. This demonstrates that even cells with extreme differentiation features can undergo direct fate conversion. Our data further suggest that the reactivation of this developmental mechanism in adults can drive tissue remodeling in human chronic airway disease, a paradigm resembling cancer formation and progression.

developmental biology↗

bioRxiv2020

Par3A and Par3B orchestrate podocyte architecture by regulating RhoA levels

Glomerular diseases are a major cause for chronic kidney disorders. In the majority of cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and subsequent loss of podocytes. Here, we establish a link between components of the Par3 polarity complex and actin regulators, which are necessary to establish and maintain the podocytes architecture utilizing both, mouse and Drosophila models. We demonstrate that the two mammalian Par3 proteins, Par3A and Par3B, share redundant functions despite differing in their ability to interact with other components of the Par complex. Only simultaneous inactivation of both Par3 proteins causes a severe disease phenotype in mouse podocytes by regulating Rho-GTP levels involving the actin regulators Synaptopodin and CD2AP in an aPKC independent manner.

cell biology↗

bioRxiv2019

Cell-type specific innervation of cortical pyramidal cells at their apical tufts

We investigated the synaptic innervation of apical tufts of cortical pyramidal cells in a region between layers 1 and 2 using 3-D electron microscopy (3D-EM) applied to four cortical regions in mouse. Across all cortices, we found the relative inhibitory input at the apical dendrites main bifurcation to be more than 3-fold stronger for layer 2 pyramidal cells than for all other pyramidal cells. Towards the distal tuft dendrites in upper layer 1, however, the relative inhibitory input was about 2-fold stronger for L5 pyramidal cells than for all others. Only L3 pyramidal cells showed homogeneous inhibitory input density. The inhibitory to excitatory synaptic balance is thus specific for the types of pyramidal cells. Inhibitory axons preferentially innervated either layer 2 or L3/5 apical dendrites, but not both. These findings describe connectomic principles for the control of pyramidal cells at their apical dendrites in the upper layers of the cerebral cortex and point to differential computational properties of layer 2, layer 3 and layer 5 pyramidal cells in cortex.

neuroscience↗

bioRxiv2018

Dense connectomic reconstruction in layer 4 of the somatosensory cortex

The dense circuit structure of the mammalian cerebral cortex is still unknown. With developments in 3-dimensional (3D) electron microscopy, the imaging of sizeable volumes of neuropil has become possible, but dense reconstruction of connectomes from such image data is the limiting step. Here, we report the dense reconstruction of a volume of about 500,000 m3 from layer 4 of mouse barrel cortex, about 300 times larger than previous dense reconstructions from the mammalian cerebral cortex. Using a novel reconstruction technique, FocusEM, we were able to reconstruct a total of 0.9 meters of dendrites and about 1.8 meters of axons investing only about 4,000 human work hours, about 10-25 times more efficient than previous dense circuit reconstructions. We find that connectomic data alone allows the definition of inhibitory axon types that show established principles of synaptic specificity for subcellular postsynaptic compartments. We find that also a fraction of excitatory axons exhibit such subcellular target specificity. Only about 35% of inhibitory and 55% of excitatory synaptic subcellular innervation can be predicted from the geometrical availability of membrane surface, revoking coarser models of random wiring for synaptic connections in cortical layer 4. We furthermore find evidence for enhanced variability of synaptic input composition between neurons at the level of primary dendrites in cortical layer 4. Finally, we obtain evidence for Hebbian synaptic weight adaptation in at least 24% of connections; at least 35% of connections show no sign of such previous plasticity. Together, these results establish an approach to connectomic phenotyping of local dense neuronal circuitry in the mammalian cortex.

neuroscience↗

bioRxiv2018

FluoEM: Virtual labeling of axons in 3-dimensional electron microscopy data for long-range connectomics

Volume electron microscopy (3D EM) has enabled the dense reconstruction of neuronal circuits in datasets that are so far about a few hundred micrometers in extent. In mammalian brains, most neuronal circuits are however highly non-local, such that a large fraction of the synapses in such a volume of neuropil originates from distant projection sources. The labeling and identification of such long-range axonal inputs from multiple sources within a densely reconstructed EM dataset has been notoriously difficult because of the limited color label space of EM. Here, we present FluoEM, a set of experimental and computational methods that allows the identification of multi-color fluorescently labeled axons in dense EM data without the need for artificially introduced fiducial marks or direct label conversion for EM. The approach is based on correlated imaging of the tissue and computational matching of neurite reconstructions, amounting to a virtual color labeling of axons in dense EM circuit data. We show that the identification of fluorescent light-microscopically (LM) imaged axons in 3D EM data from mouse cortex is faithfully possible as soon as the EM dataset is about 40-50 m in extent, relying on the unique trajectories of axons in dense mammalian neuropil. The method is exemplified for the identification of longdistance axonal input into layer 1 of the mouse cerebral cortex.

neuroscience↗

bioRxiv2017

SynEM: Automated synapse detection for connectomics

Nerve tissue contains a high density of chemical synapses, about 1 per {micro}m3 in the mammalian cerebral cortex. Thus, even for small blocks of nerve tissue, dense connectomic mapping requires the identification of millions to billions of synapses, of which about 80-90% are excitatory synapses. While the focus of connectomic data analysis has been on neurite reconstruction, synapse detection becomes limiting when datasets grow in size and dense mapping is required. Here, we report SynEM, a method for automated detection of excitatory synapses from conventionally en-bloc stained 3D electron microscopy image stacks. The approach is based on a segmentation of the image data and focuses on classifying borders between neuronal processes as synaptic or non-synaptic. SynEM yields 98% precision and recall in binary excitatory cortical connectomes with no user interaction. It scales to large volumes of cortical neuropil, plausibly even whole-brain datasets. SynEM removes the burden of manual synapse annotation for large densely mapped connectomes.

neuroscience↗