Biology subjects

Lampe, M.

Publications and source records attributed to Lampe, M..

13 recordsLinked to original sources

bioRxiv2025

Loss of class 3 PI3K complex leads to retention of Wg at the apical membrane in polarized tissue

Wnt/Wingless (Wg) signalling is a key regulator of tissue patterning and morphogenesis in Drosophila, coordinating cell fate decisions and long-range morphogen signalling. In wing imaginal discs, Wg proteins rely on specialized trafficking machinery, including the cargo receptor Evi/Wls, and are secreted via multiple routes, comprising a glypican-dependent Wg pool and an endocytosis-dependent Wg pool. However, the cellular mechanisms controlling Wg secretion and post-endocytic trafficking in Drosophila remain incompletely understood. We performed an in vivo kinome- and phosphatome-wide CRISPR-Cas9 screen in Drosophila wing imaginal discs using endogenous fluorescently tagged Wg as a readout. Genetic perturbations were combined with super-resolution microscopy and ex vivo pharmacological treatments to resolve Wg and Evi/Wls secretion dynamics. We identified Vps15, a core subunit of the class III phosphatidylinositol 3-kinase (PI3K (III)) complex, as a critical factor of Wg secretion. Loss of Vps15 caused apical accumulation of Wg in Wg-secreting cells, selectively impairing an endocytosis-dependent Wg pool while leaving a glypican-mediated Wg pool intact. In contrast to Wg, Evi/Wls did not accumulate in PI3K (III) mutant cells, but instead was subject to proteasome-dependent degradation. Super-resolution imaging further revealed frequent spatial separation of Wg and Evi/Wls in Wg-secreting cells prior to the uptake of the endocytosis-dependent Wg pool. Our study establishes PI3K (III) perturbation as a powerful approach to dissect distinct Wg secretion routes in Drosophila wing imaginal discs. We uncovered divergent post-endocytic fates of Wg and Evi/Wls upon perturbation and provided new mechanistic insight into Wg-Evi/Wls dynamics.

developmental biology↗

bioRxiv2024

Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early Gl, Cohesin-STAGl and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in Gl, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. 0ur quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.

cell biology↗

bioRxiv2024

Reflection confocal microscopy for quantitative assessment of airway surface liquid dysregulation and pharmacological rescue in cystic fibrosis under near-physiological conditions

Proper regulation of airway surface liquid (ASL) is essential for effective mucociliary clearance (MCC) in healthy airways, and ASL depletion due to deficient cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion/fluid secretion plays an important role in the pathogenesis of mucociliary dysfunction and chronic muco-obstructive lung disease in patients with cystic fibrosis (CF). The current standard for quantitative measurements of ASL height is confocal fluorescence microscopy that has the disadvantage that it requires apical addition of volume for fluorescent staining, and hence perturbation of the ASL. Therefore, our aim was to develop a method that enables studies of ASL regulation under unperturbed conditions using reflected light by confocal microscopy of primary airway epithelial cultures grown at air-liquid interface (ALI). After apical volume addition to primary tracheal mouse cultures, confocal reflection microscopy yielded comparable ASL height as confocal fluorescence microscopy on cultures of wild-type mice, and was sensitive to detect ASL depletion on cultures of {beta}ENaC-Tg mice. Under unperturbed conditions, ASL determined by confocal reflection microscopy was significantly higher in wild-type and {beta}ENaC-Tg mice compared to values obtained by confocal fluorescence microscopy. Studies in normal and CF primary human airway epithelial cultures showed that confocal reflection microscopy was sensitive to detect effects of low temperature rescue and pharmacological modulation including improvement of CFTR function by VX-809 and VX-770 in cultures from CF patients with the F508del mutation. Our results support confocal reflection microscopy as a novel sensitive technique for quantitative studies of ASL regulation and response to therapeutic intervention under unperturbed near-physiological conditions in healthy and CF airways. NEW & NOTEWORTHYMeasurement of airway surface liquid (ASL) height by confocal fluorescence microscopy is an important tool to investigate ASL dysregulation and effects of therapeutic strategies aiming at restoring ASL volume to improve mucociliary clearance and lung function in patients with cystic fibrosis. However, confocal fluorescence microscopy has the disadvantage that it requires apical addition of volume for fluorescent staining of the ASL leading to perturbation of its height and composition. Here, we developed confocal reflection microscopy as a new method that enables quantitative assessment of ASL on highly-differentiated primary airway epithelial cultures under unperturbed near-physiological conditions by detection of refracted light.

cell biology↗

bioRxiv2024

On the annotation of Paleo- and Mesoproterozoic microfossils as fungi

How old is the crown group Fungi? Inferences from phylogenetic and fossil-based studies provided far-apart age estimates ranging between 0.75 to 2.7 billion years old. One important criterion for interpreting Paleo- and Mesoproterozoic microfossils as Fungi is their uncanny morphological resemblance with extant fungi. Here, we demonstrate that bacteria exposed to environmental conditions similar to the paleoenvironmental settings where these presumed fungi lived can spontaneously transform into their protoplasts. These protoplasts exhibit morphologies corresponding to those of presumed fungal microfossils. These observations, together with microfossil chemical composition, pose a serious challenge to interpreting Paleo- and Mesoproterozoic microfossils as fungi. Based on these results, we reiterate that morphology is not a reliable indicator of the phylogeny of microfossils older than 2.5Ga.

evolutionary biology↗

bioRxiv2023

iMOUSE - Reforming the Strategy of Refinement and Reduction for indispensable laboratory animal-based studies in translational research

Considering the intricate nature of biological processes within organisms, it is undeniable that relying solely on in vitro-generated primary-cell-like cultures or organ-like products in preclinical and basic research is insufficient to replace animal-based studies fully. This limitation is particularly significant when considering the regulations enforced by legislative assemblies worldwide. The necessity of animal-based studies to approve chemicals and medications. In contradiction, European countries aim to banish animal-based studies. Therefore, we must understand the impact of the data refinement and experiment replacement strategy we will introduce here. This project aimed to revolutionize data acquisition in animal-based studies by transforming manual observation into a reliable digital process. Reliable digital data will be generated by having the potential to reduce human bias by simply reducing human interaction. Additionally, reducing human interaction will reduce the severity levels due to stress reduction, fulfilling the 3R principles. Therefore, the first goal was to develop and implement a scalable, stable, running, and remotely accessible camera-based monitor system (the iMouse solution). At the same time, the target was to develop a retrofit solution (DigiFrame) for existing home-cage systems, not interfering with the regular workflow in animal facilities. As a result, we developed a digital monitoring system, named iMouseTV platform based on existing open-source software, allowing users to observe, record, share, and review animal-based studies within the home cage anytime from anywhere, reducing the stress level for the animals. Our systems first Proof of concept ran for over two years at the LIV in Hamburg. We also investigated an effective way to reduce data generation by setting up specific zones for detecting the motion of choice (e.g., drinking, food intake). The data sets can be stored, shared, and reviewed by users and refined by algorithms aiming to recognize the dedicated motions of the animals automatically. The implementation of the ML algorithms allows the iMouse solution to recognize whether an individual mouse was drinking and for how long and store results in the annotated video file and graph format. However, the identification and continuous tracking of the species is still in progress. In conclusion, we established a scalable human-independent monitoring and recording system, which can be implemented into the existing structures of institutions and companies without changing handling processes, to monitor animals and observe them by getting reliable digital data. Moreover, it is fundamental for automatic recognition within animal-based studies based on Artificial Intelligence.

animal behavior and cognition↗

bioRxiv2022

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we designed a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.

biophysics↗

bioRxiv2021

On the reproductive mechanism of Gram-negative protocells

Bacterial protoplasts are known to reproduce independently of canonical molecular biological processes. Their reproduction is shown to be mediated entirely by the physicochemical properties of cell constituents. However, the physiochemical properties of the cell constituents are influenced by the environmental conditions like salinity, salt composition, and mechanical stresses experienced by a cell in natural environments. The influence of such environmental conditions on protoplast reproduction is seldom investigated. Here, we studied protoplast reproduction in their native environmental conditions. Contrary to the previous perceptions of protoplasts reproducing in an erratic manner, cells in our study reproduced in a defined sequence of steps. The process of their reproduction can be explained by an interplay between intracellular metabolism, the physicochemical properties of cell constituents, and the nature of cations in the growth media. We observed a minimal leakage of intracellular constituents during protoplast reproduction, suggesting an efficient reproduction. However, the efficiency of reproduction is determined by the environmental conditions. Under favorable environmental conditions, protoplasts reproduce with nearly similar efficiency to cells that possess a cell wall. In short, here we demonstrate the simplest method of cellular reproduction and the influence of environmental conditions on this process.

biophysics↗

bioRxiv2021

On the reproductive mechanisms of Gram-positive protocells

Prokaryotes are hypothesized to have evolved from more primitive protocells. Unlike present-day cells, protocells are thought to have been devoid of complex molecular biological processes. They are believed to have mediated reproduction entirely by biophysical forces under favorable environmental conditions. Despite this proposition, little is known about the actual mechanism of their reproduction. To understand the reproduction process of protocells in their native habitat, here we used a top-down approach to transform bacterial cells into a primitive lipid vesicle-like state. Given that environmental conditions are thought to have played an essential role in mediating protocell reproduction, we then studied these cells under the presumed environmental conditions of Archaean Eon Earth. Even in the absence of functioning biological processes, cells in our study reproduced in a defined sequence of steps, always leading to the formation of viable daughter cells. Their reproduction mechanism can be explained by the interaction between intracellular metabolism, physicochemical properties of cell constituents, and, most importantly, environmental conditions. Given the simplicity of this reproduction mechanism and its suitability to environmental conditions of early Earth, we propose that protocells reproduced by this process. Moreover, this method of reproduction is also in tune with the earlier theoretical propositions on protocells, the results of the top- down approach of building a minimal cell, and the paleontological record of the Achaean Eon. Our study is the first to bridge the gap between non-living systems like lipid vesicles, living cells, and the paleontology of the Archaean Eon.

biophysics↗

bioRxiv2021

Precision localization of cellular proteins with fluorescent Fab-based probes

Currently, a major technical limitation of microscopy based image analysis is linkage error - a visualization error that is measured by the distance between the cellular protein to be detected and the fluorescence emitter used for detection. With continuously improving resolution of todays (super-resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five-fold when compared to conventional staining techniques. These reagents, called Fluo-N-Fabs, consist of an antigen binding fragment of a full-length antibody (Fab/ fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo-N-Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.

cell biology↗

bioRxiv2021

On the nature of the earliest known life forms

Microfossils from the Paleoarchean Eon are the oldest known evidence of life. Despite their significance in understanding the history of life on Earth, any interpretation of the nature of these microfossils has been a point of contention among researchers. Decades of back-and-forth arguments led to the consensus that reconstructing the lifecycles of Archaean Eon organisms is the most promising way of understanding the nature of these microfossils. Here, we transformed a Gram-positive bacterium into a primitive lipid vesicle-like state and studied it under environmental conditions prevalent on early Earth. Using this approach, we successfully reconstructed morphologies and life cycles of Archaean microfossils. In addition to reproducing microfossil morphologies, we conducted experiments that spanned years to understand the process of cell degradation and how Archaean cells could have undergone encrustation minerals (in this case, salt), leading to their preservation as fossilized organic carbon in the rock record. These degradation products strongly resemble fossiliferous features from Archaean rock formations. Our observations suggest that microfossils aged between 3.8 to 2.5Ga most likely were liposome-like protocells that have evolved physiological pathways of energy conservation but not the mechanisms to regulate their morphology. Based on these observations, we propose that morphology is not a reliable indicator of taxonomy in these microfossils.

evolutionary biology↗

bioRxiv2019

Photoactivation of silicon rhodamines via a light-induced protonation

We present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophores outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

biophysics↗

bioRxiv2018

Bleaching-independent, whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores

We demonstrate bleaching-independent STED microscopy using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging in fixed and living cells. Foremost, we achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy with up to 100 min acquisition time.

biophysics↗

bioRxiv2018

A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing and dynamic localization during mitosis by fluorescence correlation spectroscopy-calibrated live cell imaging and super-resolution microscopy. While [~]35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, [~]195,000 Condensin I complexes dynamically bind in two steps, in prometaphase and early anaphase. The two Condensins rarely co-localize at the chromatid axis, where Condensin II is centrally confined but Condensin I reaches [~]50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of [~]450 kb at the chromatid axis whose size is then reduced by Condensin I binding to [~]90 kb in prometaphase and [~]70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.

cell biology↗