Biology subjects

Hahn, I.

Publications and source records attributed to Hahn, I..

11 recordsLinked to original sources

bioRxiv2026

Dynamic diffusion analysis of the yeast plasma membrane using Airyscan based microscopic techniques

The yeast plasma membrane (PM) is highly compartmentalised into distinct nanoscale domains. The mechanisms by which this organisation regulates surface proteins are not fully understood, and it remains unclear how different biophysical modalities capture diffusion kinetics across varying spatial scales. Using confocal microscopy and an Airyscan2 detector, we benchmarked two prominent techniques: Fluorescence Correlation Spectroscopy (FCS) via the Zeiss Dynamics Profiler and Fluorescence Recovery After Photobleaching (FRAP). We quantified the lateral diffusion of three functionally diverse GFP-tagged model proteins: the exocytic t-SNARE Sso2, the lipid-binding protein Pmp3, and the eisosome-associated protein Ycp4. While diffusion coefficients aligned tightly between both modalities for Pmp3 and Ycp4, Sso2 exhibited a stark 14-fold discrepancy, displaying drastically faster local mobility by FCS compared to macroscopic recovery by FRAP. High-resolution 3D Structured Illumination Microscopy (3D-SIM) shows that Sso2 is partitioned into regional subdomains, that occupy less PM area than the network-like localisation of Pmp3. Our findings suggest that FCS captures rapid, localised diffusion within these microenvironments, whereas FRAP measures highly restricted transit across domain boundaries. Ultimately, this work demonstrates that membrane diffusion coefficients cannot be interpreted in isolation and capturing true lateral mobility requires pairing kinetic measurements with super-resolution spatial mapping to decode complex membrane compartmentalisation.

cell biology↗

bioRxiv2025

Steady-State Visually Evoked Potentials as Readouts for Abnormal GSK3β Activity in Drosophila

Glycogen synthase kinase 3{beta} (GSK3{beta}) is important in neuronal development and maintenance, acting as a key regulator by controlling a broad range of cellular processes. The effects of its dysregulation range from impairments in axonal transport and energy metabolism to synapse formation and neuronal plasticity. However, how such cellular defects link to neuronal dysfunction is less well studied despite the links between GSK3{beta} and various neurological conditions, such as Alzheimers and Parkinsons diseases, mood disorders and autism. Here we used a steady-state visually evoked potential (SSVEP) assay with Drosophila to measure the effect of altering GSK3{beta} expression on neuronal function. We recorded SSVEPs from flies that expressed constitutively active or inactive (kinase-dead) variants of GSK3{beta} and showed that the visual responses of these flies differed from those of controls. This was indicated by increased response latency and reduced photoreceptor maximum response (Rmax). Importantly, multivariate pattern classification can distinguish between over- and inactive GSK3{beta} conditions. Taken together, we show that SSVEPs provide a powerful tool for future studies to link the molecular and cellular functions of GSK3{beta} with their effects on neuronal function. Furthermore, we introduce a monitoring protocol that ensures the quality of datasets to support future fly SSVEP experiments.

neuroscience↗

bioRxiv2025

Microtubule curling as an efficient readout to uncover fundamental concepts of axonal cell biology

Nerve fibres (aka axons) are the slender, up-to-meter-long processes of nerve cells that wire nervous systems. These delicate structures must survive for an organisms lifetime, making them prime lesion sites in neurodegeneration. Their long-term maintenance requires the homeostasis of complex local cell biology upheld by motor protein-driven transport along microtubule bundles that run uninterrupted along axons. To gain an understanding of axonal homeostasis, we report and discuss here the functional loss of 105 genes from a wide range of cell biological processes using a standardised Drosophila primary neuron system. [~]40% of these gene deficiencies caused microtubule bundle disturbances referred to as microtubule-curling, which we use as an indicator of axonal atrophy. Our live imaging showed that microtubule-curling initiates in areas where axons widen, such as areas close to the soma, branch points or growth cones. In wild-type neurons, any initiated curling was contained, but it persisted as growing footprints in mutant curl-promoting conditions. Closer analyses of 20 curl-promoting conditions suggested a general classification into two groups: mutations affecting axonal physiology cause ROS-mediated microtubule-curling, whereas mutations causing motor hyperactivation or affecting microtubule-regulating proteins cause structurally induced ROS-independent curling. As will be discussed, our data provide consistent support for the previously proposed dependency cycle of local axon homeostasis model which can explain the long-standing conundrum that genes from a wide range of cell biological processes often have mutational links to the same class of inherited neurodegenerative disease.

cell biology↗

bioRxiv2025

Spectraplakin cooperates with noncentrosomal microtubule regulators to orient dendritic microtubules in Drosophila

The differential microtubule organization in axons and dendrites underlies neuronal polarity and developmental processes like neurite pruning. How neurons achieve their specific microtubule organization during development is an area of active research, and it has been especially hard to explain how dendritic microtubules are oriented with their plus ends towards the soma. Transient microtubule nucleation from tips of early growing dendrites has been detected in some systems and would explain how orientation is set up. In a survey for cytoskeletal regulators involved in dendrite pruning and microtubule organization in Drosophila, we found the spectraplakin Short stop (Shot), an actin/microtubule crosslinker. Loss of Shot causes microtubule orientation defects already during early dendrite development, when Shot is transiently recruited to tips of growing dendrites via its actin binding domain. Genetic and functional evidence suggest that Shots primary function in this process is to locally stabilize microtubules. We also provide evidence for a developmentally transient microtubule nucleation mechanism. Our data highlight the importance of transient and localized microtubule regulation for dendritic microtubule organization.

developmental biology↗

bioRxiv2025

Neuronal differentiation enhances a cytoplasmic pool of tousled-like kinase 2 (TLK2)

Mental retardation autosomal dominant 57 (MRD57) is a rare neurodevelopmental disorder characterised by delayed language and psychomotor development, intellectual disability, hypotonia, gastrointestinal issues and facial dysmorphia. It is linked to genetic mutations in the serine/threonine kinase TLK2, characterised by haploinsufficiency and in some cases, its loss or impaired kinase function. TLK2 is an established cell cycle regulator that has been extensively studied in mitotic cells. It is upregulated in cancers, driving tumour growth, however, the role of TLK2 in postmitotic neurons is not understood. We therefore aimed to gain insight into how TLK2 mutations cause MRD57 by determining where TLK2 is expressed in the brain and its subcellular localisation during neuronal differentiation. Public human and mouse brain transcriptomic data revealed splice variant diversity in the N-terminus of TLK2, which contains its nuclear localisation sequence (NLS). Using splice-specific in situ hybridisation probes, we observed expression of TLK2 isoforms that contain and lack the NLS in the mouse hippocampus and cerebellum. We confirmed these findings in human SH-SY5Y neuroblastoma cells, and found that neuronal differentiation of these cells enhances a cytoplasmic pool of TLK2 by two mechanisms: nuclear export of full length TLK2 and increased expression of TLK2 splice variants lacking the NLS. Finally, acute stimuli that mimic synaptic activity were sufficient to elicit nuclear export of TLK2. Our data highlight the need to establish the neuronal cytoplasmic substrates of TLK2 and determine how the loss of TLK2 activity in MRD57 might impact their function in the developing and mature brain.

neuroscience↗

bioRxiv2024

Mis-regulation of GSK-3β causes axonal microtubule curling through Shot and Tau

Glycogen Synthase Kinase 3{beta} (GSK-3{beta}) is a key coordinator of neuronal development and maintenance; hyperactive GSK-3{beta} is linked to neurodevelopmental and -degenerative diseases and therefore a promising therapeutic target. In neurons, GSK-3{beta} coordinates the cytoskeleton by phosphorylating microtubule-binding proteins. In this study, we found that tight regulation of GSK-3{beta} kinase activity is required for the maintenance of parallel microtubule bundles in Drosophila and rat axons. Up- or down-regulation of GSK-3{beta} led to axons forming pathological swellings in which microtubule bundles disintegrated into disorganised, curled microtubules. We identified the microtubule bundling proteins Shot and Tau as key GSK-3{beta} targets and found that GSK-3{beta} exerted its regulatory effect on microtubule bundling through them. GSK-3{beta} regulates the ability of Shot and Tau to attach to microtubules and/or the plus-end protein Eb1. Mis-regulation of GSK-3{beta} leads to the loss of Eb1-Shot-mediated guidance of polymerising microtubules into parallel bundles, thus causing disorganisation. We propose microtubule disorganisation as a new explanation for how GSK-3{beta} hyperactivity leads to neurodegeneration and why global inhibition of GSK-3{beta} has not been successful in clinical trials for neuronal disorders.

cell biology↗

bioRxiv2021

Re-evaluating the actin-dependence of spectraplakin functions during axon growth and maintenance

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require bundles of microtubules that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and axon growth inhibition. The underlying mechanisms are best understood for Drosophila Short stop (Shot) and believed to involve cytoskeletal cross-linkage: the N-terminal calponin homology (CH) domains bind to F-actin, and the C-terminus to microtubules and Eb1. Here we have gained new understanding by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shots CH domains induces changes in Shot function - with a Lifeact-containing Shot variant causing remarkable remodelling of neuronal microtubules. In addition to actin-MT cross-linkage, we find strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. We argue that these likely involve the neuronal Shot-PH isoform, which is characterised by a large, unexplored central plakin repeat region (PRR). Work on PRRs might therefore pave the way towards important new mechanisms of axon biology and architecture that might similarly apply to central PRRs in mammalian spectraplakins.

neuroscience↗

bioRxiv2020

Tau, XMAP215/Msps and Eb1 jointly regulate microtubule polymerisation and bundle formation in axons

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each others localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet size, comet number and comet velocity, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies and how to treat them. Summary statementEb1, XMAP215 and tau co-operate interdependently in axons to promote the polymerisation of microtubules and their organisation into the parallel bundles required for axonal transport.

cell biology↗

bioRxiv2019

The model of local axon homeostasis - explaining the role and regulation of microtubule bundles in axon maintenance and pathology

Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brain and body. In spite of their challenging morphology, they usually need to be maintained for an organisms lifetime. This makes them key lesion sites in pathological processes of ageing, injury and neurodegeneration. The morphology and physiology of axons crucially depends on the parallel bundles of microtubules (MTs), running all along to form their structural backbones and highways for life-sustaining cargo transport and organelle dynamics. Understanding how these bundles are formed and then maintained will provide important explanations for axon biology and pathology. Currently, much is known about MTs and the proteins that bind and regulate them, but very little about how they functionally integrate to regulate axons. As an attempt to bridge this important knowledge gap, we explain here the model of local axon homeostasis, based on our own experiments and published data. (1) As the default, we observe that axonal MTs have a strong bias to become disorganised, likely caused by the physical forces imposed by motor proteins and their life-sustaining functions during intra-axonal transport and dynamics. (2) Preventing MT disorganisation and promoting their bundled conformation, requires complex machinery involving most or even all major classes of MT-binding and - regulating proteins. As will be discussed, this model offers new explanations for axonopathies, in particular those linking to MT-regulating proteins and motors; it will hopefully motivate more researchers to study MTs, and help to decipher the complex regulatory networks that can explain axon biology and pathology.

cell biology↗

bioRxiv2018

Efa6 regulates axon growth, branching and maintenance by eliminating off-track microtubules at the cortex

Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the Arf activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones, they enhance axon growth, in axon shafts, explorative MTs cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.\n\nSummary statementThe cortical collapse factor Efa6 inhibits microtubule polymerising outside axonal bundles. Thereby it limits axon growth and branching, but preserves microtubule bundle organisation crucial for axon maintenance.

cell biology↗

bioRxiv2017

Drosophila Short stop as a paradigm for the role and regulation of spectraplakins

Spectraplakins are evolutionarily well conserved cytoskeletal linker molecules that are true members of three protein families: plakins, spectrins and Gas2-like proteins. Spectraplakin genes encode at least 7 characteristic functional domains which are combined in a modular fashion into multiple isoforms, and which are responsible for an enormous breadth of cellular functions. These functions are related to the regulation of actin, microtubules, intermediate filaments, intracellular organelles, cell adhesions and signalling processes during the development and maintenance of a wide variety of tissues. To gain a deeper understanding of this enormous functional diversity, invertebrate genetic model organisms, such as the fruit fly Drosophila, can be used to develop concepts and mechanistic paradigms that can inform the investigation in higher animals or humans. Here we provide a comprehensive overview of our current knowledge of the Drosophila spectraplakin Short stop (Shot). We describe its functional domains and isoforms and compare them with those of the mammalian spectraplakins dystonin and MACF1. We then summarise its roles during the development and maintenance of the nervous system, epithelia, oocytes and muscles, taking care to compare and contrast mechanistic insights across these functions in the fly, but especially also with related functions of dystonin and MACF1 in mostly mammalian contexts. We hope that this review will improve the wider appreciation of how work on Drosophila Shot can be used as an efficient strategy to promote the fundamental concepts and mechanisms that underpin spectraplakin functions, with important implications for biomedical research into human disease.

cell biology↗