Biology subjects

Mueller-Reichert, T.

Publications and source records attributed to Mueller-Reichert, T..

7 recordsLinked to original sources

bioRxiv2026

Geometric optics organizes organelle interactions and positioning

Microtubule asters are central to positioning organelles and organizing intracellular architectures. We show that the multi-stage choreography of microtubule asters which positions pronuclei during the first cell division in Caenorhabditis elegans, is governed by a cellular analog of geometric optics. Large-scale electron tomography reveals that astral microtubules reach cortical and pronuclear surfaces largely along line-of-sight trajectories, leaving complementary regions shadowed and inaccessible. Laser ablation identifies surface-anchored motors pulling on microtubules as the dominant drivers of motion. We develop a biophysical model incorporating dynamic terminator curves that separate microtubule-accessible and -inaccessible surfaces, and quantitatively recapitulate aster separation, pronuclear migration, centering, and rotation in control and genetically perturbed embryos. More broadly, robust positioning emerges from a feedback loop wherein intracellular geometry gates microtubule access, thus sculpting the distribution and magnitude of pulling forces, which in turn reshape that geometry.

cell biology↗

bioRxiv2025

Fluorescence Lifetime Unmixing: A New Workflow for FLIM Live-Cell Imaging

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime unmixing, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we unmixed the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we unmixed fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-FLIM-phasor-plotter to implement conversion, visualization, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

cell biology↗

bioRxiv2024

In situ quantification of ribosome number by electron tomography

Ribosomes, discovered in 1955 by George Palade, were originally described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, numerous studies were conducted on both ribosome structure and function. The basic question, however, how many ribosomes are contained within whole cells has not been addressed so far. Here we developed a microscopic method to determine the total number of ribosomes in human hTRET-RPE-1 and in nematode cells from different tissues in Caenorhabditis elegans hermaphrodites. Electron tomography of high-pressure frozen, freeze substituted and resin-embedded samples revealed that the number of ribosomes as determined by this electron tomographic approach closely matches the biochemical estimates for the hTRET-RPE-1 cells. In addition, RPOA-1-depleted worms compared to control worms showed an expected decrease in the number of ribosomes in two out of three different tissue types investigated. The application of our imaging-based approach to directly count the number of ribosomes in given samples will significantly enhance our basic understanding of ribosome localization and distribution within cells and tissues.

cell biology↗

bioRxiv2024

Setting up an institutional OMERO environment for bioimage data: perspectives from both facility staff and users

Modern bioimaging core facilities at research institutions are essential for managing and maintaining high-end instruments, providing training and support for researchers in experimental design, image acquisition and data analysis. An important task for these facilities is the professional management of complex multi-dimensional bioimaging data, which are often produced in large quantity and very different file formats. This article details the process that led to successfully implementing the OME Remote Objects system (OMERO) for bioimage-specific research data management (RDM) at the Core Facility Cellular Imaging (CFCI) at the Technische Universitat Dresden (TU Dresden). Ensuring compliance with the FAIR (findable, accessible, interoperable, reusable) principles, we outline here the challenges that we faced in adapting data handling and storage to a new RDM system. These challenges included the introduction of a standardized group-specific naming convention, metadata curation with tagging and Key-Value pairs, and integration of existing image processing workflows. By sharing our experiences, this article aims to provide insights and recommendations for both individual researchers and educational institutions intending to implement OMERO as a management system for bioimaging data. We showcase how tailored decisions and structured approaches lead to successful outcomes in RDM practices.

bioinformatics↗

bioRxiv2023

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Like other volume electron microscopy approaches, Automated Tape Collecting Ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. ATUM-SEM of serial semi-thick sections and consecutive ET of selected re-mounted sections combines SEMs fast target recognition and coarse rendering capability with high-resolution volume visualizations, respectively, thus enabling multi-scale interrogation of cellular ultrastructure. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. This workflow is a seamless zoom-in on structural BBB pathology from the micro- to the nanometer scale. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

neuroscience↗

bioRxiv2021

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

biophysics↗

bioRxiv2019

Soluble tubulin is locally enriched at mitotic centrosomes in C. elegans

During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymer tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimer and polymer at mitotic centrosomes in early C. elegans embryos. We show that a C. elegans one-cell stage centrosome at metaphase contains more than ten thousand microtubules with a total polymer concentration of 230 M. Centrosomes concentrate soluble /{beta} tubulin by about tenfold over the cytoplasm, reaching peak values of 470 M, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 M. These findings support in vitro data suggesting that microtubule nucleation in C. elegans centrosomes is driven in part by concentrating soluble tubulin.

cell biology↗