Biology subjects

Wiesner, T.

Publications and source records attributed to Wiesner, T..

11 recordsLinked to original sources

bioRxiv2026

Calcium transient detection and segmentation with the astronomically motivated algorithm for background estimation and transient segmentation (Astro-BEATS)

SignificanceFluorescence-based Ca2+-imaging is a powerful tool for studying localized neuronal activity, including miniature Synaptic Calcium Transients, providing real-time insights into synaptic activity. These transients induce only subtle changes in the fluorescence signal, often barely above baseline, which poses a significant challenge for automated synaptic transient detection and segmentation. AimDetecting astronomical transients similarly requires efficient algorithms that will remain robust over a large field of view with varying noise properties. We leverage techniques used in astronomical transient detection for miniature Synaptic Calcium Transient detection in fluorescence microscopy. ApproachWe present Astro-BEATS, an automatic miniature Synaptic Calcium Transient segmentation algorithm that incorporates image estimation and source-finding techniques used in astronomy and designed for Ca2+-imaging videos. Astro-BEATS uses the Rolling Hough Transform filament detector to construct an estimate of the expected (transient-free) fluorescence signal of both the dendritic foreground and the background. Subtracting this baseline signal yields difference images displaying transient signals. We use Density-Based Spatial Clustering of Applications with Noise to find sources clustered in spatial and temporal space. ResultsAstro-BEATS outperforms current threshold-based approaches for synaptic Ca2+ transient detection and segmentation. The produced segmentation masks can be used to train a supervised deep learning algorithm for improved synaptic Ca2+ transient detection in Ca2+-imaging data. The speed of Astro-BEATS and its applicability to previously unseen datasets without re-optimization makes it particularly useful for generating training datasets for deep learning-based approaches. ConclusionAstro-BEATS greatly reduces the time needed for the annotation of synaptic Ca2+ transient and removes the significant overhead of human expert annotation, enabling consistent analysis of new Ca2+-imaging datasets.

bioinformatics↗

bioRxiv2025

Non-synaptic exocytosis along the axon shaft and its regulation by the submembrane periodic skeleton

Neuronal communication relies on signaling molecules transferred via exo- and endocytosis throughout the brain. Historically, studies have focused on vesicle exo- and endocytosis and their release machinery at synapses, and much less is known about non-synaptic exocytosis. If and how vesicles can access the plasma membrane along the axonal shaft, overcoming the insulating layer of the membrane-associated periodic scaffold, remains unclear. Here, we used fast live-cell imaging of mature cultured hippocampal neurons expressing the vamp2-pHluorin reporter to map sponta-neous exocytosis along axons. We detected non-synaptic exocytic events along the axon shaft that concentrated at the axon initial segment. Perturbation of the membrane-associated actin-spectrin skeleton revealed its role in regulating shaft exocytosis, similarly to its recently demonstrated role in shaping axonal endocytosis. To visualize the nanoscale arrangement of exocytic locations, we developed a novel correlative live-cell/two - color, single-molecule localization microscopy (SMLM) approach. We observed that regions of exocytosis are devoid of the submembrane spectrin mesh, with these spectrin-free areas being spatially separated from the spectrin clearings that contain clathrin-coated pits. Overall, our work reveals a new process of spontaneous exocytosis along the axon shaft, and how the axonal submem-brane skeleton shapes a heterogeneous landscape that uniquely segregates vesicular trafficking events.

cell biology↗

bioRxiv2024

Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model

Chemical synapses are fundamental units for the transmission of information throughout the nervous system. The cyto-skeleton allows to build, maintain and transform both pre- and postsynaptic contacts, yet its organization and the role of its unique synaptic nanostructures are still poorly understood. Here we present a presynapse-on-glass model where presynaptic specializations are robustly induced along the axons of cultured neurons by micropatterned dots of neuroligin, allowing the controlled orientation and easy optical visualization of functional induced presynapses. We demonstrate the relevance and usefulness of this presynapse-on-glass model for the study of presynaptic actin architecture, showing that a majority of induced presynapses are enriched in actin, with this enrichment being correlated to higher synaptic cycling activity. We confirm our previous results on bead-induced presynapses by identifying the same distinct actin nanostructures with-in presynapses: corrals, rails and mesh. Furthermore, we leverage the controlled orientation of the presynapse-on-glass model, visualizing the arrangement of these actin structures relative to the active zone nanoclusters using multicolor 3D Single Molecule Localization Microscopy (SMLM), and relative to the sub-diffractive localization exocytic events using a correlative live-cell and SMLM approach.

neuroscience↗

bioRxiv2024

Quantitative Analysis of Miniature Synaptic Calcium Transients Using Positive Unlabeled Deep Learning

Ca2+ imaging methods are widely used for studying cellular activity in the brain, allowing detailed analysis of dynamic processes across various scales. Enhanced by high-contrast optical microscopy and fluorescent Ca2+ sensors, this technique can be used to reveal localized Ca2+ fluctuations within neurons, including in sub-cellular compartments, such as the dendritic shaft or spines. Despite advances in Ca2+ sensors, the analysis of miniature Synaptic Calcium Transients (mSCTs), characterized by variability in morphology and low signal-to-noise ratios, remains challenging. Traditional threshold-based methods struggle with the detection and segmentation of these small, dynamic events. Deep learning (DL) approaches offer promising solutions but are limited by the need for large annotated datasets. Positive Unlabeled (PU) learning addresses this limitation by leveraging unlabeled instances to increase dataset size and enhance performance. This approach is particularly useful in the case of mSCTs that are scarce and small, associated with a very small proportion of the foreground pixels. PU learning significantly increases the effective size of the training dataset, improving model performance. Here, we present a PU learning-based strategy for detecting and segmenting mSCTs. We evaluate the performance of two 3D deep learning models, StarDist-3D and 3D U-Net, which are well established for the segmentation of small volumetric structures in microscopy datasets. By integrating PU learning, we enhance the 3D U-Nets performance, demonstrating significant gains over traditional methods. This work pioneers the application of PU learning in Ca2+ imaging analysis, offering a robust framework for mSCT detection and segmentation. We also demonstrate how this quantitative analysis pipeline can be used for subsequent mSCTs feature analysis. We characterize morphological and kinetic changes of mSCTs associated with the application of chemical long-term potentiation (cLTP) stimulation in cultured rat hippocampal neurons. Our data-driven approach shows that a cLTP-inducing stimulus leads to the emergence of new active dendritic regions and differently affects mSCTs subtypes.

bioinformatics↗

bioRxiv2024

TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

cancer biology↗

bioRxiv2024

Spectrin condensates provide a nidus for assembling the periodic axonal structure

Coordinated assembly of individual components into higher-order structures is a defining theme in biology, but underlying principles are not well-understood. In neurons, /{beta} spectrins, adducin, and actinfilaments assemble into a lattice wrapping underneath the axonal plasma membrane, but mechanistic events leading to this periodic axonal structure (PAS) are unclear. Visualizing PAS components in axons as they develop, we found focal patches in distal axons containing spectrins and adducin (but sparse actin filaments) with biophysical properties reminiscent of biomolecular condensation. Overexpressing spectrin-repeats - constituents of /{beta}-spectrins - in heterologous cells triggered condensate formation, and preventing association of {beta}II-spectrin with actin-filaments/membranes also facilitated condensation. Finally, overexpressing condensate-triggering spectrin repeats in neurons before PAS establishment disrupted the lattice, presumably by competing with innate assembly, supporting a functional role for biomolecular condensation. We propose a condensation-assembly model where PAS components form focal phase-separated condensates that eventually unfurl into a stable lattice-structure by associating with subplasmalemmal actin. By providing local depots of assembly parts, biomolecular condensation may play a wider role in the construction of intricate cytoskeletal structures.

cell biology↗

bioRxiv2023

pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis

Exocytosis is a fundamental process used by eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we have developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons. Summary StatementExocytosis is an essential process by which cells change shape, alter membrane composition, and communicate with other cells. Though all eukaryotic cells carry out exocytosis, the regulation of vesicle fusion, the cargo of vesicles, and the role exocytosis plays in cell fate differ greatly across cell types. Here, we developed a flexible and robust set of tools to enable automatic identification and analysis of exocytic events across a wide range of cell types, vesicle types, and imaging conditions.

cell biology↗

bioRxiv2021

Task-Assisted GAN for Resolution Enhancement and Modality Translation in Fluorescence Microscopy

AbstractWe introduce a deep learning model that predicts super-resolved versions of diffraction-limited microscopy images. Our model, named Task- Assisted Generative Adversarial Network (TA-GAN), incorporates an auxiliary task (e.g. segmentation, localization) closely related to the observed biological nanostructures characterization. We evaluate how TA-GAN improves generative accuracy over unassisted methods using images acquired with different modalities such as confocal, brightfield (diffraction-limited), super-resolved stimulated emission depletion, and structured illumination microscopy. The generated synthetic resolution enhanced images show an accurate distribution of the F-actin nanostructures, replicate the nanoscale synaptic cluster morphology, allow to identify dividing S. aureus bacterial cell boundaries, and localize nanodomains in simulated images of dendritic spines. We expand the applicability of the TA-GAN to different modalities, auxiliary tasks, and online imaging assistance. Incorporated directly into the acquisition pipeline of the microscope, the TA-GAN informs the user on the nanometric content of the field of view without requiring the acquisition of a super-resolved image. This information is used to optimize the acquisition sequence, and reduce light exposure. The TA-GAN also enables the creation of domain-adapted labeled datasets requiring minimal manual annotation, and assists microscopy users by taking online decisions regarding the choice of imaging modality and regions of interest.

bioinformatics↗

bioRxiv2020

Neuronal activity remodels the F-actin based submembrane lattice in dendrites but not axons of hippocampal neurons

The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves Ca2+ and NMDA-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.

neuroscience↗

bioRxiv2020

LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

Merkel cell carcinoma (MCC) is a highly aggressive, neuroendocrine skin cancer that is either associated with the clonal integration of the Merkel cell polyomavirus or with chronic sun exposure1,2. Immunotherapy is initially effective in many patients with metastatic MCC, but the response is rarely durable3,4. MCC lacks actionable mutations that could be utilized for targeted therapies, but epigenetic regulators, which govern cell fate, provide unexplored therapeutic entry points. Here, we performed a pharmacological screen in MCC cells, targeting epigenetic regulators. We discovered that the lysine-specific histone demethylase 1A (LSD1/KDM1A) is required for MCC growth in vitro and in vivo. HMG20B (BRAF35), a poorly characterized subunit of the LSD1-CoREST complex, is also essential for MCC proliferation. LSD1 inhibition in MCC disrupts the LSD1-CoREST complex, directly induces the expression of key regulators of the neuronal lineage and of members of the TGF{beta} pathway, and activates a gene expression signature corresponding to normal Merkel cells. Our results provide a rationale for evaluating LSD1 inhibitors, which are currently being tested in patients with leukemia and solid tumors, in MCC.

cancer biology↗

bioRxiv2018

B cells sustain inflammation and improve survival in human melanoma

Tumor associated inflammation is one of important predictors of response to immune checkpoint blockade. Understanding molecular processes that regulate tumor inflammation is key to improve the efficacy of checkpoint blockade. Established mechanisms that underlie therapy response and resistance have centered on anti-tumor T cell responses.\n\nWe show that tumor-associated B cells are vital to T cell functions. They promote recruitment of CD8+ T cells through plasmablast-like cells with expression of pro- and anti-inflammatory factors. Plasmablast-like cells are associated with improved survival of patients with metastatic melanoma and their response to checkpoint blockade. Plasmablast-like B cells express chemokines for T cell-attraction. Depletion of tumor-associated B cells by anti-CD20 immunotherapy of metastatic melanoma patients causes a remarkable decrease in tumor CD8+ T cells. These findings indicate that tumor-associated B cells orchestrate and sustain tumor inflammation, recruit CD8+ T effector cells and are key to therapeutic response and patients overall survival.

cancer biology↗