Biology subjects

Heinrich, L.

Publications and source records attributed to Heinrich, L..

10 recordsLinked to original sources

bioRxiv2025

Arp2/3 complex contributes to the actin-dependent uptake of Aspergillus terreus conidia by alveolar epithelial cells

Aspergillus terreus is an opportunistic fungal pathogen associated with high mortality rates and intrinsic resistance to amphotericin B. Its ability to persist within host tissues without inducing strong immune responses was suggested to contribute to poor clinical outcomes. The cellular mechanisms underlying A. terreus interactions with host cells remain largely unexplored. In this study, we have used a micropattern-based infection model to investigate the early interactions between A. terreus conidia and alveolar epithelial cells, focusing on the role of Arp2/3-dependent actin remodeling. This system allows quantitative analysis of conidia-cell interactions under defined spatial conditions. We show that A. terreus conidia rapidly bind to micropatterned A549 cell islands, with conidial numbers increasing over time. Conidia were found in actin- and Lamp1-positive vesicles already after one hour of infection. Inhibition of the Arp2/3 complex significantly impaired conidial binding and disrupted the formation of actin-positive vesicles, confirming the essential role of Arp2/3-mediated actin remodeling in early stages of conidial uptake. A subset of internalized conidia was localized to Lamp1-positive phagolysosomes and accumulated over time. Interestingly, we have identified a small but consistent population of phagolysosomes decorated with actin patches, potentially resembling actin flashes. These structures were entirely abolished upon Arp2/3 inhibition, indicating active cytoskeletal remodeling at the phagolysosomal interface. Our findings provide the first mechanistic insights into A. terreus internalization by alveolar epithelial cells and establish Arp2/3-mediated actin dynamics as a key process in early host-pathogen interactions. This cellular pathway may contribute to intracellular persistence and help understand the delayed onset of A. terreus infections.

microbiology↗

bioRxiv2024

Biological Boundary Conditions Regulate the Internalization of Aspergillus Fumigatus Conidia by Alveolar Cells

IntroductionThe lung environment is defined by unique biological boundary conditions, including complex alveolar geometry, extracellular matrix composition and mechanical forces generated during respiration. These factors were shown to regulate alveolar permeability, surfactant secretion, cell contractility and apoptosis, but their role in fungal infections remains unknown. Aspergillus fumigatus is a critical fungal pathogen that causes severe pulmonary infections in immunocompromised individuals. Our study addresses a knowledge gap by investigating how boundary conditions affect A. fumigatus conidia interactions with alveolar epithelial cells. MethodsWe applied micropatterned substrates to confine cells into defined shapes and densities, allowing precise control over geometric conditions and extracellular matrix composition. Using cell line stably expressing the phagolysosomal protein Lamp1-NeonGreen and multiplane fluorescent microscopy, we evaluated A. fumigatus conidia binding and internalization efficiency. ResultsWe observed significantly faster and more efficient A. fumigatus conidia internalization in cells confined on micropatterns compared to previously reported studies using cell monolayers. Altering cell geometry, density, and extracellular matrix composition strongly affected conidia binding and localization to Lamp1+ phagolysosomes. Cells on X-shaped or multicellular micropatterns showed higher internalization rates, particularly at the periphery, suggesting spatial heterogeneity in pathogen uptake. Additionally, changes in extracellular matrix composition influenced the intracellular trafficking of A. fumigatus conidia. DiscussionOur findings emphasize the essential role that local mechanical and biochemical cues play in shaping the interactions between fungal pathogens and alveolar cells. Understanding how lung boundary conditions change in disease states will provide important insights into fungal infection outcomes.

microbiology↗

bioRxiv2023

Selection of optimal cell lines for high-content phenotypic screening

High-content microscopy offers a scalable approach to screen against multiple targets in a single pass. Prior work has focused on methods to select "optimal" cellular readouts in microscopy screens. However, methods to select optimal cell line models have garnered much less attention. Here, we provide a roadmap for how to select the cell line or lines that are best suited to identify bioactive compounds and their mechanism of action (MOA). We test our approach on compounds targeting cancer-relevant pathways, ranking cell lines in two tasks: detecting compound activity ("phenoactivity") and grouping compounds with similar MOA by similar phenotype ("phenosimilarity"). Evaluating six cell lines across 3214 well-annotated compounds, we show that optimal cell line selection depends on both the task of interest (e.g. detecting phenoactivity vs. inferring phenosimilarity) and distribution of MOAs within the compound library. Given a task of interest and set of compounds, we provide a systematic framework for choosing optimal cell line(s). Our framework can be used to reduce the number of cell lines required to identify hits within a compound library and help accelerate the pace of early drug discovery.

pharmacology and toxicology↗

bioRxiv2022

Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signaling, and lipid transfer. Using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometer proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization and mobility. These findings indicate that desmosomes and the keratin cytoskeleton pattern the distribution of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction. One-Sentence SummaryThe desmosome adhesive junction regulates the organization and dynamics of the endoplasmic reticulum network.

cell biology↗

bioRxiv2022

Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology

BackgroundHuman induced pluripotent stem cell (iPSC) models have been hailed as a breakthrough for understanding disease and developing new therapeutics. The major advantage of iPSC-derived neurons is that they carry the genetic background of the donor, and as such could be more predictive for clinical translation. However, the development of these cell models is time-consuming and expensive and it is thus critical to maximize biomarker readout from every model that is developed. One option is to use a highly multiplexed biomarker imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ at single-cell resolution. New MethodThis paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs. ResultsWe differentiated human iPSCs into mixed neuronal and glial cultures on glass coverslips. We then developed and optimized a panel of 21 antibodies to phenotype iPSC-derived neuronal subtypes of cortical, dopaminergic, and striatal neurons, as well as astrocytes, and pre-and postsynaptic proteins. Comparison with existing methodsCompared to standard immunocytochemistry, CODEX oligoconjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing. ConclusionWe show that CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology. Furthermore, we demonstrate both cellular and subcellular resolution imaging of multiplexed biomarkers in the same samples. CODEX is a powerful technique that complements other single-cell omics technologies for in-depth phenotype analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/479039v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@68d49dorg.highwire.dtl.DTLVardef@5ea424org.highwire.dtl.DTLVardef@16a763forg.highwire.dtl.DTLVardef@7950fc_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOC_FLOATNO CODEX(R) Multiplex Imaging in human iPSC neurons [A-D] Schematic drawings of the tools and steps used for Co-Detection by indEXing (CODEX) imaging. [A] Target-specific antibodies are conjugated to unique DNA oligonucleotide barcodes. Fluorescent reporter (excitation wavelength at 488 nm, 550 nm, or 647 nm/Cy5) carrying the complementary DNA (to the barcode) enables barcode-specific binding of the reporter to the antibody and detection by fluorescence microscopy. [B] Neuronal cell cultures are prepared for the CODEX staining and imaging by several fixation steps with different PFA concentrations followed by incubation with 100% acetone. Residual acetone is removed by drying the sample. After rehydration with PBS, autofluorescence is quenched by exposure to broad-spectrum LED light. Following a pre-staining fixation step, the sample is incubated with a mix of all conjugated primary antibodies. Excessive, unbound antibodies are removed by a washing step, leaving only the bound antibodies followed by a final post-staining fixation. [C] The CODEX Instrument Manager performs the multicycle run and controls the microscope software for automated addition of reporters, imaging, and washing of the samples (pre-stained with primary antibodies) to remove reporters from each cycle. After imaging, bound reporters are removed without damaging the tissue using a solvent, and the next set of reporters (conjugated to different barcodes) are added. [D] CODEX(R) Processor processes raw files and performs stitching, deconvolution, background subtraction, and cell segmentation. The processed images can be viewed and analyzed with the CODEX(R) Multiplex Analysis Viewer (MAV) plugin using Fiji software. C_FIG

neuroscience↗

bioRxiv2020

Automatic whole cell organelle segmentation in volumetric electron microscopy

Cells contain hundreds of different organelle and macromolecular assemblies intricately organized relative to each other to meet any cellular demands. Obtaining a complete understanding of their organization is challenging and requires nanometer-level, threedimensional reconstruction of whole cells. Even then, the immense size of datasets and large number of structures to be characterized requires generalizable, automatic methods. To meet this challenge, we developed an analysis pipeline for comprehensively reconstructing and analyzing the cellular organelles in entire cells imaged by focused ion beam scanning electron microscopy (FIB-SEM) at a near-isotropic size of 4 or 8 nm per voxel. The pipeline involved deep learning architectures trained on diverse samples for automatic reconstruction of 35 different cellular organelle classes - ranging from endoplasmic reticulum to microtubules to ribosomes - from multiple cell types. Automatic reconstructions were used to directly quantify various previously inaccessible metrics about these structures, including their spatial interactions. We show that automatic organelle reconstructions can also be used to automatically register light and electron microscopy images for correlative studies. We created an open data and open source web repository, OpenOrganelle, to share the data, computer code, and trained models, enabling scientists everywhere to query and further reconstruct the datasets.

cell biology↗

bioRxiv2019

Automatic Detection of Synaptic Partners in a Whole-Brain Drosophila EM Dataset

The study of neural circuits requires the reconstruction of neurons and the identification of synaptic connections between them. To scale the reconstruction to the size of whole-brain datasets, semi-automatic methods are needed to solve those tasks. Here, we present an automatic method for synaptic partner identification in insect brains, which uses convolutional neural networks to identify post-synaptic sites and their pre-synaptic partners. The networks can be trained from human generated point annotations alone and require only simple post-processing to obtain final predictions. We used our method to extract 244 million putative synaptic partners in the fifty-teravoxel full adult fly brain (FAFB) electron microscopy (EM) dataset and evaluated its accuracy on 146,643 synapses from 702 neurons with a total cable length of 312 mm in four different brain regions. The predicted synaptic connections can be used together with a neuron segmentation to infer a connectivity graph with high accuracy: between 92% and 96% of edges linking connected neurons are correctly classified as weakly connected (less than five synapses) and strongly connected (at least five synapses). Our synaptic partner predictions for the FAFB dataset are publicly available, together with a query library allowing automatic retrieval of up- and downstream neurons.

neuroscience↗

bioRxiv2019

Different α2δ Accessory Subunits Regulate Distinctly Different Aspects of Calcium Channel Function in the Same Drosophila Neurons

Voltage gated calcium channels (VGCCs) regulate neuronal excitability and translate activity into calcium dependent intracellular signaling. The pore forming 1 subunit of high voltage activated (HVA) VGCCs operates not in isolation but associates with 2{delta} accessory subunits. 2{delta} subunits can affect calcium channel biophysical properties, surfacing, localization and transport, but their in vivo functions are incompletely understood. In vertebrates, it is largely unknown whether different combinations of the four 2{delta} and the 7 1 subunits mediate different or partially redundant functions or whether different 1/2{delta} combinations regulate different aspects of VGCC function. This study capitalizes on the relatively simpler situation in the Drosophila genetic model that contains only two genes for HVA calcium channels, one Cav1 homolog and one Cav2 homolog, both with well-described functions in different compartments of identified motoneurons. We find that both d2{delta}1 and d2{delta}3 (stj) are broadly but differently expressed in the nervous system. Both are expressed in motoneurons, but with differential subcellular localization. Functional analysis reveals that d2{delta}3 is required for normal Cav1 and Cav2 current amplitudes and for correct Cav2 channel function in all neuronal compartments, axon terminal, axon, and somatodendritic domain. By contrast, d2{delta}1 does not affect Cav1 or Cav2 current amplitudes or presynaptic function, but it is required for correct Cav2 channel allocation to the axonal versus the dendritic domain. Therefore, different 2{delta} subunits are required in the same neurons to precisely regulate distinctly different functions of HVA calcium channels, which is in accord with specific 2{delta} mutations causing different brain diseases.\n\nSignificance StatementCalcium influx through the pore forming 1-subunit of voltage gated calcium channels serves essential neuronal functions, such as synaptic vesicle release, control of action potential shape and frequencies, synaptic input computations, and transcriptional control. Localization and function of 1-calcium channel subunits depend on interactions with 2{delta} accessory subunits. Here we present in vivo analysis of Drosophila motoneurons revealing that different 2{delta} subunits independently regulate distinctly different aspects of calcium channel function in the same neuron, such as current amplitude and dendritic versus axonal channel localization. Our findings start unraveling how different 1/2{delta} combinations regulate functional calcium channel diversity in different sub-neuronal compartments, and may provide an entry point toward understanding how mutations of different 2{delta} genes underlie brain diseases.

neuroscience↗

bioRxiv2018

An unbiased template of the Drosophila brain and ventral nerve cord

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individuals neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time.

neuroscience↗

bioRxiv2017

Effects of population- and seed bank noise on neutral evolution and efficacy of natural selection

Population genetics models typically consider a fixed population size and a unique selection coefficient. However, population dynamics inherently generate noise in numbers of individuals and selection acts on various components of the individuals fitness. In plant species with seed banks, the size of both the above- and below-ground compartments present noise depending on seed production and the state of the seed bank. We investigate if this noise has consequences on 1) the rate of genetic drift, and 2) the efficacy of selection. We consider four variants of two-allele Moran-type models defined by combinations of presence and absence of noise in above-ground and seed bank compartments. Time scale analysis and dimension reduction methods allow us to reduce the corresponding Fokker-Planck equation to a one-dimensional diffusive Moran model. We first show that if the above-ground noise classically affects the rate of genetic drift, below-ground noise reduces the diversity storage effect of the seed bank. Second, we consider that selection can act on four different components of the plant fitness: plant or seed death rate, seed production or seed germination. Our striking result is that the efficacy of selection for seed death rate or germination rate is reduced by seed bank noise, whereas selection occurring on plant death rate or seed production is not affected. We derive the expected site-frequency spectrum reflecting this heterogeneity in selection efficacy between genes underpinning different plant fitness components. Our results highlight the importance to consider the effect of ecological noise to predict the impact of seed banks on neutral and selective evolution.

evolutionary biology↗