Biology subjects

Reber, S.

Publications and source records attributed to Reber, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The physical state of the nucleus tracks its function across eukaryotes

The nucleus is consistently less dense than the surrounding cytoplasm, a difference proposed to arise from selective nuclear import. The functional significance of low nuclear density, however, remains unknown. Because density is a bulk property of the cell, acute perturbations inevitably have pleiotropic consequences. We therefore turned to the natural experiments offered by the tree of life where nuclei vary in genome size, chromatin organisation, import selectivity and transcriptional state, and asked whether low nuclear density is tied to particular nuclear architectures or functions. Using dinoflagellates, Giardia and ciliates, we show nuclear density is uncoupled from genome size. In ciliates, two nuclei in one cytoplasm reach opposite densities relative to it, governed by nuclear import rather than transcription. We thus confirm nuclear import as a major determinant of nuclear density and reveal nuclei that break the low-density rule.

biophysics↗

bioRxiv2026

Protein-driven colloid-osmotic pressure controls nuclear size, organization, and function

The size of the cell nucleus is tightly controlled and changes in nuclear size correlate with altered nuclear function during development, cell differentiation and senescence. How nuclear size is regulated and whether changes thereof are functionally relevant remains unclear. Here, we demonstrate that nuclear size is determined by the osmotic pressure exerted by proteins in yeast, human cells and frog egg extracts. The biophysical model we establish solves the long-standing question of how the nuclear-to-cytoplasmic ratio is regulated and maintained. Furthermore, altering protein-driven osmotic balance modulates the physical properties of the nucleus, with a direct effect on chromatin organization and gene expression: Nuclear enlargement causes the dissolution of heterochromatic structures and derepression of subtelomeres and transposons, while simultaneously down-regulating highly expressed housekeeping genes. Importantly, these global transcriptional patterns closely mimic the gene expression changes that occur as yeast, human, and drosophila cells enlarge. Importantly, artificially forcing nuclear compression is sufficient to reverse these size-associated expression changes. Together, our findings provide a quantitative, mechanistic explanation for the coupling between the size of the nucleus and the cell, and they establish nuclear size as a modulator of chromatin organization and gene expression.

cell biology↗

bioRxiv2026

Weak interactions drive selective proteome demixing and tune the differential response to environmental perturbations

The intracellular space is a crowded environment where macromolecules perform distinct tasks despite pervasive "non-specific" interactions. Whether these interactions are functionally relevant and how they influence cellular organization remains unclear. Here, we developed QuPID-MS, which measures the propensity of proteins to phase separate in native cell extracts proteome-wide. We find that weak interactions drive condensation of half of the proteome in a crowding- and temperature-dependent manner and we present evidence that this proteome demixing occurs in cells. Importantly, protein condensation properties are conserved and broadly change when cells adapt to new environments, demonstrating that weak interactions are regulated and linked to function. Indeed, condensation of the growth regulator TORC1 coincides with its rapid inactivation, while high solubility of the stress-activated Hog1 ensures its activity across conditions. We thus uncover a fundamental organizing principle that allows tuning of cell growth to environmental fluctuations while ensuring other processes function robustly despite perturbations.

cell biology↗

bioRxiv2026

Inhomogeneous Tau polymerization, core-shell organization, and seed formation during Tau condensate aging

Several proteins aggregating in neurodegenerative diseases spontaneously segregate into liquid condensates, which can catalyze protein aggregation. How liquid-solid transitions are catalyzed in the confined condensate volume is not clear. For the microtubule associated protein Tau, aggregating intra-neuronally in Alzheimer's disease, we show that, during maturation, Tau condensates convert into elastic protein networks, accompanied by inhomogeneous polymerization of the condensate interior and the formation of high-density nodes and a 'shell'. During condensation, Tau molecules extend, favoring intermolecular interactions and priming for progressive parallel Tau arrangement that can enable amyloid-like Tau oligomerization. In cells, aged condensates seed small Tau clusters in cytosol and at the nuclear envelope, precursors of larger aggregates. By bridging molecular to condensate level, we present mechanistic insight into how Tau condensates evolve into pathological, beta-structure containing seeds. The interior of aged Tau condensates remains accessible for smaller molecules, providing the opportunity to molecularly target Tau seed formation inside condensates.

biochemistry↗

bioRxiv2026

Synthetic data enables human-grade microtubule analysis with foundation models for segmentation

Studying microtubules (MTs) and their mechanical properties is central to understanding intracellular transport, cell division, and drug action. While important, experts still need to spend many hours manually segmenting these filamentous structures. The suitability of state-of-the-art methods for this task cannot be systematically assessed, as large-scale labeled datasets are missing. We address this gap by presenting the synthetic dataset SynthMT, produced by tuning a novel image generation pipeline on real-world interference reflection microscopy (IRM) frames of in vitro reconstituted MTs without requiring human annotations. In our benchmark, we evaluate nine fully automated methods for MT analysis in both zero- and Hyperparameter Optimization (HPO)-based few-shot settings. Across both settings, classical algorithms and current foundation models still struggle to achieve the accuracy required for biological downstream analysis on in vitro MT IRM images that humans perceive as visually simple. However, a notable exception is the recently introduced SAM3 model. After HPO on only ten random SynthMT images, its text-prompted version SAM3Text achieves near-perfect and in some cases super-human performance on unseen, real data. This indicates that fully automated MT segmentation has become feasible when method configuration is effectively guided by synthetic data. To enable progress, we publicly release the dataset, the generation pipeline, and the evaluation framework at DATEXIS.github.io/SynthMT-project-page. Author summaryUnderstanding the behavior of microtubules -- stiff filaments inside cells -- is essential for studying fundamental cell biological processes and for developing therapies for diseases such as cancer and neurodegenerative conditions. Yet, analyzing microtubule images is slow and labor-intensive, as researchers must manually trace these thin, overlapping filaments, which can take hours and is prone to errors. We therefore asked whether current fully automated segmentation methods are ready to replace manual microtubule analysis, and how synthetic data can be used to rigorously evaluate and improve them. To address these questions, we created a synthetic dataset that mimics real microtubule images by capturing the appearance and variability of real microscopy. Importantly, this dataset can be generated without any manual annotations. Using this dataset, we evaluated a range of segmentation methods and found that most of them struggled to accurately identify filaments. However, we discovered that a recent foundation model, when guided by a simple text instruction and tuned on only a few synthetic images, can achieve near-perfect, human-level performance on previously unseen real microtubule imaging data. Our work demonstrates that fully automated microtubule analysis is now possible and provides a reproducible framework that other researchers can use to evaluate and improve their methods. This opens the door to faster, more consistent studies of microtubules, ultimately accelerating discoveries in cell biology and therapeutic research.

bioinformatics↗

bioRxiv2025

A validated panel of commercial antibodies for reliable detection of FET proteins.

The FET protein family comprises the highly conserved RNA-binding proteins FUS, EWS and TAF15 which are implicated in RNA metabolism and neurodegenerative diseases such as ALS and FTLD. Despite their structural similarity, reliable detection of individual FET proteins remains challenging due to antibody cross-reactivity and inconsistent localisation patterns reported in the literature. To address this, we systematically evaluated 23 commercially available antibodies for specificity and performance across western blotting, immunocytochemistry, and immunohistochemistry. Using single and double shRNA knockdowns in HeLa cells, we confirmed target engagement and identified antibodies which were specific as well as those with significant cross-reactivity. Immunofluorescence in rat primary neurons and HeLa cells showed antibody and cell type dependent variations in nuclear and cytoplasmic localisation; from these, we identified a subset that demonstrated high-quality, region-specific staining in postmortem human brain tissue. Our findings highlight substantial variability in antibody performance and underscore the need for rigorous validation to ensure reproducibility in FET protein research. We present a validated panel of antibodies suitable for diverse applications, providing a critical resource for studies of FET protein biology and pathology.

neuroscience↗

bioRxiv2025

Adaptations in Plasmodium tubulin determine unique microtubule architectures, mechanics and drug susceptibility

Microtubules are ubiquitous yet diverse cytoskeleton filaments. However, tubulin conservation presents challenges in understanding the origins of diverse microtubule architectures. The mechanisms by which microtubule architecture varies through the life cycle of the malaria-causing parasite Plasmodium are not understood and provide a valuable framework for exploring how intrinsic properties of tubulin contribute to architectural variety. Using parasite-purified tubulin, we determined structures of P. falciparum microtubules by cryo-electron microscopy. Parasite-specific sequences change the tubulin dimer structure, thereby modifying drug susceptibility and polymer mechanical properties. Within the P. falciparum microtubule, lateral contacts are smaller but stronger, and the lattice is stiffer than in mammalian microtubules. Non-canonical microtubule architectures found in parasites are highly similar to those observed in vitro, validating the physiological relevance of these properties. Our findings show how evolutionary adaptation of tubulin modulates the material properties of the microtubule cytoskeleton.

biophysics↗

bioRxiv2025

Hierarchical Heterogeneities in Spatio-Temporal Dynamics of the Cytoplasm

The physical properties of the cytoplasm fundamentally constrain the dynamics and fidelity of cellular processes. Endogenous fluctuations carry signatures of these properties, yet how their collective dynamics across mesoscopic spatiotemporal scales relate to cytoplasmic heterogeneity and organization remains poorly understood. Here, we use differential dynamic microscopy (DDM) to quantify these fluctuations in eukaryotic cytoplasms. In Xenopus laevis egg extracts, we identify signatures of subdiffusive fractional Brownian motion (fBm) and non-Gaussian displacement distributions, consistent with a crowded, heterogeneous environment. We introduce an empirical model combining fBm with an inverse Gaussian diffusivity distribution, enabling robust estimation of fractional diffusivities and scaling exponents across conditions. Denser cytoplasm exhibits lower fractional diffusivity and scaling exponent, while energy addition induces non-stationary dynamics. Stabilization of microtubule networks introduces a secondary timescale, rationalized by a two-state fBm model separating cytosolic and network-associated contributions. In HeLa cells, the framework reveals comparable scaling exponents but lower diffusivities than in egg extract. Upon microtubule depolymerization, the dynamics become slower yet more Brownian-like. This highlights dual roles of the cytoskeleton as confinement but also as fluidizer of the cytoplasm. These results establish label-free DDM as an interpretable, collective, multiscale readout of how composition, activity, and cytoskeletal organization shape hierarchical cytoplasmic dynamics.

biophysics↗

bioRxiv2024

Mechanistic basis of temperature-adaptation in microtubule dynamics across frog species

Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. We thus lack a mechanistic framework that links functional adaptability with environmental pressures. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures: we combine in vitro reconstitution assays, quantitative biochemistry, and cryogenic electron microscopy to show how a small number of primary sequence changes influences the energy landscape of tubulin interactions and thereby mediates cold-adaptation and microtubule stability. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.

biochemistry↗

bioRxiv2024

Cell State-Specific Cytoplasmic Material Properties Control Spindle Architecture and Scaling

Mitotic spindles are dynamically intertwined with the cytoplasm they assemble in. How the physicochemical properties of the cytoplasm affect spindle architecture and size remains largely unknown. Using quantitative biochemistry in combination with adaptive feedback microscopy, we investigated mitotic cell and spindle morphology during neural differentiation of embryonic stem cells. While tubulin biochemistry and microtubule dynamics remained unchanged, spindles changed their scaling behaviour: in differentiating cells, spindles were significantly smaller than those in equally-sized undifferentiated stem cells. Integrating quantitative phase imaging, biophysical perturbations and theory, we found that as cells differentiated, their cytoplasm became more dilute. The concomitant decrease in free tubulin activated CPAP (centrosomal P4.1-associated protein) to enhance the centrosomal nucleation capacity. As a consequence, in differentiating cells, microtubule mass shifted towards spindle poles at the expense of the spindle bulk, explaining the differentiation-associated switch in spindle architecture. This study shows that cell state-specific cytoplasmic density tunes mitotic spindle architecture. Thus, we reveal physical properties of the cytoplasm as a major determinant in organelle size control.

cell biology↗

bioRxiv2023

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes

The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

biophysics↗

bioRxiv2023

Single cell RNA sequencing in isogenic FUS and TARDBP mutant ALS lines reveals early mitochondrial dysfunction as a common pathway in motor neurons

Mutations in the RNA/DNA-binding proteins FUS and TDP-43 cause the fatal disease amyotrophic lateral sclerosis (ALS). The precise mechanisms behind the selective motor neuron degeneration remain unclear and it is uncertain if ALS-causative mutations trigger motor neuron death through shared or distinct pathogenic pathways. To address these two questions, we performed single-cell RNA sequencing across neuron types derived from isogenic induced pluripotent stem cell lines, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. The mutations elicited 5- to 15-fold greater transcriptional responses in motor neurons than interneurons. Approximately 20% of transcripts uniquely dysregulated in motor neurons were shared across FUS mutations, with half being driven by FUS gain-of-function. Among these, a majority pointed towards mitochondrial impairments, with attenuated pathways shared with the TARDBP M337V mutation. Meta-analysis demonstrated convergence on mitochondrial dysfunction with C9orf72-ALS patient-derived motor neurons. We observed impaired mitochondrial motility across ALS motor axons, even in isogenic FUS R244C motor neurons, which retain FUS in the nucleus, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early signs of mitochondrial dysfunction unique to motor neurons could have profound implications for their survival and represent promising therapeutic targets across multiple ALS forms.

neuroscience↗

bioRxiv2022

Predicting disordered regions driving phase separationof proteins under variable salt concentration

We investigate intrinsically disordered regions (IDRs) of phase separating proteins regarding their impact on liquid-liquid phase separation (LLPS) of the full protein. Our theoretical approach uses a mean-field theory that accounts for sequence-dependent electrostatic interactions via a random-phase approximation (RPA) and in addition allows for variable salt concentration for the condensed and dilute protein phases. The numerical solution of the complete phase diagrams together with the tie lines that we derive for this model system leaves two parameters to be determined by fitting experimental data on concentrations of all species involved in the system. For our comparisons, we focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that its long IDR near the C-terminus promotes LLPS, which we validate through direct comparison with in vitro experimental results under the same physiological conditions. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS has been intensively studied. Apart from the LC domain we here investigate theoretically two IDRs, one near the N-terminus and another near the C-terminus. Our theoretical analysis of these domains predict that the IDR at the N-terminus (aa 1-285) is the main driver of LLPS of FUS by comparison to in vitro experiments of the full length protein under the same physiological temperature and salt conditions. SIGNIFICANCEIntrinsically disordered proteins are drivers of cellular liquid-liquid phase separation. However, it remains a challenge to directly predict the phase behaviour of a protein based on its primary sequence, and under physiological conditions. We present a random-phase approximation that allows for variable salt concentration and thus accounts for salt partitioning. We use this to link the sequence of the disordered regions with the behaviour of the complete protein through direct comparisons to in vitro phase-separation assays. In particular, for FUS we determine the exact region responsible for LLPS, weighting in a long-standing debate.

biophysics↗

bioRxiv2022

Calcitic prisms of the giant seashell Pinna nobilis form light guide arrays

The shells of the Pinnidae family are based on a double layer of single-crystal-like calcitic prisms and inner aragonitic nacre, a structure known for its outstanding mechanical performance. However, on the posterior side, shells are missing the nacreous layer, which raises the question of whether there could be any functional role in giving up this mechanical performance. Here, we demonstrate that the prismatic part of the Pinna nobilis shell exhibits unusual optical properties, whereby each prism acts as an individual optical fiber guiding the ambient light to the inner shell cavity by total internal reflection. This pixelated light channeling enhances both spatial resolution and contrast while reducing angular blurring, an apt combination for acute tracking of a moving object. Our findings may offer insights into the evolutionary aspects of light-sensing and imaging by biological materials and introduce a conceptual framework for the development of bio-inspired multifunctional ceramics and architectured light-tracking materials.

biophysics↗

bioRxiv2022

Barrier-properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We found that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier, all consistent with a sieve-like phase. Phase separation tolerated mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remained preserved. NTR-entry, however, was sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we found arginines to promote FG-phase-entry apparently also by hydrophobic interactions/ H-bonding and not just through cation-{pi} interactions. Although incompatible with NTR{middle dot}cargo complexes, a YG phase displayed remarkable transport selectivity, particularly for engineered GFPNTR-variants. GLFG to FSFG mutations made the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxed this hypercohesion. Thus, antagonism between cohesion and NTR{middle dot}FG interactions is key to transport selectivity.

biophysics↗

bioRxiv2021

Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While {beta}-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

biochemistry↗

bioRxiv2021

Volumetric morphometry reveals mitotic spindle width as the best predictor of spindle scaling

The function of cellular structures at the mesoscale is dependent on their geometry and proportionality to cell size. The mitotic spindle is a good example why length and shape of intracellular organelles matter. Spindle length determines the distance over which chromosomes will segregate and spindle shape ensures bipolarity. While we still lack a systematic and quantitative understanding of subcellular morphometrics, new imaging techniques and volumetric data analysis promise novel insights into scaling relations across different species. Here, we introduce Spindle3D, an open-source plug-in that allows for the quantitative, unbiased, and automated analysis of 3D fluorescent data of spindles and chromatin. We systematically analyse different cell types, including somatic cells, stem cells and one-cell embryos across different phyla to derive volumetric relations of spindle, chromatin, and cell volume. Taken together, our data indicate that mitotic spindle width is a robust indicator of spindle volume, which correlates linearly with chromatin and cell volume both within single cell types and across metazoan phyla.

systems biology↗

bioRxiv2019

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

molecular biology↗