Biology subjects

Rotkopf, R.

Publications and source records attributed to Rotkopf, R..

13 recordsLinked to original sources

bioRxiv2026

Chromatin tethering to the nuclear envelope enhances its accessibility to RNAPII and promotes chromatin asymmetric organization

The intrinsic tendency of chromatin to self-attract competes with its association with RNA Polymerase II (RNAPII), a prerequisite for efficient transcription. Using high-resolution live imaging of chromatin and RNAPII organization in Drosophila larval muscle nuclei, we demonstrate that chromatin tethering to the nuclear envelope via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is essential for maintaining chromatin three-dimensional organization. Disruption of chromatin-lamina interactions either in LINC mutants or following knockdown of Barrier-to-Autointegration Factor (BAF) results in enhanced chromatin clustering in the nucleoplasm and reduced RNAPII-chromatin interaction. Consistent with these observations, computer simulations revealed an inverse relationship between the chromatin cluster size and the degree of chromatin tethering to the nuclear lamina. We also measured chromatin distribution with respect to the nuclear lamina and found asymmetry of RNAPII distributions within chromatin clusters, which correlated with their proximity to the nuclear envelope, a relationship that is lost in nuclei lacking a functional LINC complex. Our findings demonstrate that chromatin association with the nuclear envelope counteracts chromatin self-attraction and facilitates RNAPII binding to DNA.

molecular biology↗

bioRxiv2025

Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution

Gene amplification in the form of extrachromosomal DNA (ecDNA) is a frequent driver in multiple cancer types. As ecDNA lack centromeres, their mitotic segregation does not follow traditional inheritance principles. However, the mechanisms that govern ecDNA fate following mitosis remain unclear. We found that ecDNA undergo numerical and structural optimization under increased selective pressure, with mitotic chromosomal tethering, or detachment, dictating ecDNA fate. When tethered, ecDNA aggregates promote uneven distribution into the newly formed daughter cells, thereby driving inter-cellular numerical heterogeneity and rapid increase of amplification under selective pressure. Mitotically detached ecDNA frequently encapsulate within micronuclei of variable size and content that appear to be highly fragile. Strikingly, ecDNA enclosed in very small micronuclei, which we term nanonuclei, are being actively degraded through autophagy. Together with ongoing structural rearrangements, nanonuclear ecDNA degradation promotes their structural evolution, which facilitates cancer cell adaptation. Our work highlights ecDNA aggregation, micronucleation, and degradation, as pivotal events in directing cancer genome evolution trajectories.

cancer biology↗

bioRxiv2025

Comparative developmental transcriptomics of Drosophila mushroom body neurons highlights the mevalonate pathway as a regulator of axon growth

The ability of neurons to extend axons is governed by tightly regulated genetic programs that vary across developmental stages and cell types. Understanding the molecular features that control axon growth potential is critical for uncovering how neural circuits form, mature, and respond to injury or disease. The Drosophila mushroom body (MB) offers a powerful model to dissect axon growth programs, as lineage-related Kenyon cells (KCs) undergo different developmental events under shared spatiotemporal conditions. During metamorphosis, {gamma}-KCs undergo axon pruning, followed by developmental regrowth at the same time-frame as /{beta}-KCs initiate axon growth - thus providing a unique opportunity to compare these distinct growth paradigms. We thus performed RNA-sequencing of /{beta}-and {gamma}-KCs during their initial growth and developmental regrowth, respectively, revealing dynamic transcriptional changes and identifying 300 shared genes upregulated during both growth states. A targeted loss-of-function screen revealed genes specifically required for either /{beta} initial growth, {gamma} regrowth, or both. Focusing on one such candidate, Pmvk, we found that it plays a crucial role in axon regrowth by acting within the mevalonate pathway. Notably, other enzymes in this pathway were also required, suggesting that the entire metabolic pathway is essential for supporting regrowth. Genetic mutant analyses and rescue exepriements suggest that Pmvk likely controls axon regrowth via Rheb, an effector of the TOR pathway, which we previously found to be required for regrowth. Our developmental transcriptomic atlas not only advances understanding of intrinsic axon growth programs, but also provides candidate genes and a valuable framework for future studies aimed at enhancing axon regeneration in the adult nervous system.

neuroscience↗

bioRxiv2024

A Blueprint of Sex-Specific Neuronal Regulation in the C. elegans Nervous System at Single-Cell Resolution

Sex-specific behaviors are often attributed to differences in neuronal wiring and molecular composition, yet how genetic sex shapes the molecular architecture of the nervous system at the individual neuron level remains unclear. Here, we use single-cell RNA sequencing to profile the transcriptome of sex-shared neurons in adult Caenorhabditis elegans males and hermaphrodites. We uncover widespread molecular dimorphism across the nervous system, including in previously unrecognized neuron-types such as the touch receptors. Neuropeptides and signaling-related genes exhibit strong sex-biased expression, particularly in males, reinforcing the notion that neuropeptides are crucial for diversifying connectome outputs. Despite these differences, neurotransmitter identities remain largely conserved, indicating that functional dimorphism arises through modulatory, not identity-defining, changes. We show that sex-biased expression of neurotransmitter-related genes correlates with bias in outgoing synaptic connectivity and identify regulatory candidates for synaptic wiring, including both shared and sex-specific genes. This dataset provides a molecular framework for understanding how subtle regulatory differences tune conserved circuits to drive sex-specific behaviors.

neuroscience↗

bioRxiv2024

Wnt/β-Catenin coordinates muscle spindle development by regulating capsule differentiation, intrafusal fiber nuclei aggregation and proprioceptive nerve endings

Proprioception is essential for the regulation of posture, movement, and musculoskeletal integrity. The muscle spindle, a mechanosensory organ composed of multiple specialized tissues, detects stretch and provides proprioceptive feedback. Despite its importance, the molecular mechanisms that orchestrate the development of the spindle components remain poorly understood. Here, we reveal the involvement of the Wnt/{beta}-catenin pathway in muscle spindle development. We show that Wnt ligands and their Frizzled receptors are expressed in the developing spindle and that {beta}-catenin is active throughout development in capsule cells and in bag2 intrafusal fibers. Embryonic deletion of {beta}-catenin from capsule and intrafusal fibers induced widespread transcriptomic changes, which led to significant malformations, including impaired capsule cell differentiation, disrupted nuclear organization in intrafusal fibers, and disorganized proprioceptive nerve endings. Postnatal deletion of {beta}-catenin from intrafusal fibers resulted in abnormal nerve endings and impaired proprioceptive function, indicating that the development of proprioceptive afferents is regulated by {beta}-catenin through a non-cell-autonomous mechanism acting in bag2 fibers. Collectively, our findings position the Wnt/{beta}-catenin pathway as a central regulator that acts through both cell-autonomous and non-cell-autonomous mechanisms to coordinate the development of the various spindle tissues into a functional organ.

developmental biology↗

bioRxiv2024

Elucidating early intestinal stem cell response to bacterial infection

Intestinal stem cells (ISCs) are the regenerative force of the gut epithelium. Lgr5+-ISC have been shown to respond to changes in their microenvironment by coping with different metabolites, adapting to caloric changes, and recovering from injury and inflammation. However, how pathogenic bacteria affect adult stem cell regeneration and, as a consequence, the overall tissue adaption to infection has yet to be explored in depth. Here, we interrogated early Lgr5+ ISC responses to an enteric intracellular pathogen by profiling individual IECs from the mouse small intestine. Utilizing GFP-labeled Salmonella enterica, we isolated intracellular invaded cells to elucidate invasion programs of epithelial cell subsets. In particular, we identified a Salmonella-specific infection signature comprised of antimicrobial peptide (AMP) genes, including the Defensin gene family. Our findings demonstrate that Salmonella enterica targets differentiated Paneth, enterocytes, and stem/progenitor cells at these early stages of infection. In response, a rapid Lg5+ ISC-driven cellular remodeling to enterocyte and Paneth lineages expressing AMP genes is initiated to combat the intruders. Importantly, we uncovered an ISC differentiation program via inflammasome activation to protect the crypt environment, while eliminating infected stem cells from the overall stem cell pool. This novel Lgr5+ stem cell defense mechanism not only protects the gut epithelium from persistent bacterial infection but also promotes tissue regeneration. We propose epithelial remodeling to AMP-secreting cells as a novel innate immune response to handle different gut stresses mediated by Lgr5+ ISCs to maintain organizational principles of gut homeostasis and physiology.

immunology↗

bioRxiv2023

High-energy demand and nutrient exhaustion in MTCH2 knockout cells

Mitochondrial carrier homolog 2 (MTCH2) is a regulator of apoptosis, mitochondrial dynamics, and metabolism. Loss of MTCH2 results in mitochondrial fragmentation, an increase in whole-body energy utilization, and protection from diet-induced obesity. We now show using temporal metabolomics that MTCH2 deletion results in a high ATP demand, an oxidized environment, a high lipid/amino acid/carbohydrate metabolism, and in the decrease of many metabolites. Lipidomics analyses show a strategic adaptive decrease in membrane lipids and an increase in storage lipids in MTCH2 knockout cells. Importantly, all the metabolic changes in the MTCH2 knockout cells were rescued by MTCH2 re-expression. Interestingly, this imbalance in energy metabolism and reductive potential triggered by MTCH2-deletion inhibits adipocyte differentiation, an energy consuming reductive biosynthetic process. In summary, loss of MTCH2 results in an increase in energy demand that triggers a catabolic and oxidizing environment, which fails to fuel the anabolic processes during adipocyte differentiation.

cell biology↗

bioRxiv2022

Phylogeny and biogeography of the algal DMS-releasing enzyme

Phytoplankton produce the volatile dimethyl sulfide (DMS), an important infochemical, which is emitted to the atmosphere and affecting the global climate. Albeit the enzymatic source for DMS in eukaryotes was elucidated, namely a DMSP lyase (DL) called Alma1, we still lack basic knowledge regarding its taxonomy and biogeographic distribution. We defined unique sequence motifs which enable the identification of DL homologs (DLHs) in model systems and environmental populations. We used these motifs to predict DLHs in diverse algae by analyzing hundreds of genomic and transcriptomic sequences from model systems under stress conditions and from environmental samples. Our findings show that the DL enzyme is more taxonomically widespread than previously thought, as it is encoded by known algal taxa as haptophytes and dinoflagellates, but also by chlorophytes, pelagophytes and diatoms, which were conventionally considered to lack the DL enzyme. By exploring the Tara Oceans database, we showed that DLHs are widespread across the oceans and are predominantly expressed by dinoflagellates. Certain dinoflagellate DLHs were differentially expressed between the euphotic and mesopelagic zones, suggesting a functional specialization and an involvement in the metabolic plasticity of mixotrophic dinoflagellates. In specific regions as the Southern Ocean, DLH expression by haptophytes and diatoms was correlated with environmental drivers such as nutrient availability. The expanded repertoire of putative DL enzymes from diverse microbial origins and geographic niches suggests new potential players in the marine sulfur cycle and provides a foundation to study the cellular function in marine microbes.

microbiology↗

bioRxiv2020

TENT4A poly(A) polymerase regulates translesion DNA synthesis and is mutated in endometrial cancer

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here we focus on its multilayer regulation of translesion DNA synthesis (TLS), in which DNA lesions are bypassed by error-prone DNA polymerases. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase {eta} and RAD18 E3 ligase, which guides the polymerase to replication stalling sites, and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD, and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase {eta}, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

molecular biology↗

bioRxiv2019

In vivo imaging of myonuclei during spontaneous muscle contraction reveals non-uniform nuclear mechanical dynamics in Nesprin/klar mutants

Muscle contractions produce reiterated cytoplasmic mechanical variations, which impact the nuclear membrane and potentially influence nuclear mechanotransduction. It is unclear, however, whether the mechanical dynamics of individual myonuclei changes during each contractile wave, and whether mutants whose connection to the cytoskeleton is impaired are subjected to different mechanical input.\n\nTo monitor nuclear mechanical dynamics in vivo, we imaged myonuclei along muscles during multiple spontaneous muscle contractile events, within live and intact Drosophila wild-type or Nesprin/klar mutant larvae. The data were subsequently analyzed and quantified aiming to reveal potential changes in the mechanical parameters of nuclear dynamics during muscle contraction in the Nesprin/klar mutant. Our results show that all myonuclei in control larvae exhibited comparable dynamics in the course of multiple contractile events. In contrast, myonuclei of homozygous mutant larvae lacking the Nesprin-like gene klar displayed differential dynamics relative to wild type, and higher variability between myonuclei at distinct positions along individual myofibers. Estimation of the drag force applied on individual myonuclei revealed that force fluctuations in time were considerably higher in the Nesprin/klar mutant myonuclei relative to control, reflecting a significant variability in the mechanical dynamics of individual myonuclei during contractile waves. The variable mechanical dynamics along the muscle fiber and the higher variance of Nesprin/klar mutant myonuclei may lead to altered nuclear mechanotransduction. Since mutations in Nesprin genes lead to devastating muscle and cardiac human diseases, our findings may provide new insight into the mechanism underlying these pathologies.

biophysics↗

bioRxiv2019

Robo2 regulates synaptic oxytocin content by affecting actin state

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT), and used the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were surrounded by polymerized actin. Perturbation of actin filaments by cytochalasin-D resulted in decreased synaptic OXT levels. Live imaging of the actin probe, Lifeact-EGFP, showed reduced mobility in OXT synapses in robo2 mutants, which displayed decreased synaptic OXT content. Using a novel transgenic reporter line allowing real-time monitoring of OXT-loaded vesicles, we showed that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Robo2-Cdc42 signalling, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.

developmental biology↗

bioRxiv2018

Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP

Emiliania huxleyi is a bloom forming microalga that impacts the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide (DMS). Top-down regulation of E. huxleyi blooms is attributed to viruses and grazers, however, the possible involvement of algicidal bacteria in bloom demise is still elusive. We isolated from a North Atlantic E. huxleyi bloom a Roseobacter strain, Sulfitobacter D7, which exhibited algicidal effects against E. huxleyi upon co-culturing. Both the alga and the bacterium were found to co-occur during a natural E. huxleyi bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying alga-bacterium interaction in the oceans. During interaction, Sulfitobacter D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which E. huxleyi strains that exuded higher amounts of DMSP were more susceptible to Sulfitobacter D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in a resistant algal strain to the bacterial pathogen. This DMSP-dependent pathogenicity was highly specific as compared to supplementation of propionate and glycerol. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions, as a mediator of bacterial virulence that may regulate E. huxleyi blooms.

microbiology↗

bioRxiv2017

Unmasking cellular response of a bloom-forming alga to virus infection by resolving expression profiling at a single-cell level

Marine viruses are major evolutionary and biogeochemical drivers of microbial life in the ocean. Host response to viral infection typically includes virus-induced rewiring of metabolic network to supply essential building blocks for viral assembly, as opposed to activation of anti-viral host defense. Nevertheless, there is a major bottleneck to accurately discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we use Emiliania huxleyi, a bloom-forming alga and its specific virus (EhV), as one of the most ecologically important host-virus model system in the ocean. Using automatic microfluidic setup to capture individual algal cells, we quantified host and virus gene expression on a single-cell resolution during the course of infection. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and uncover a yet unrecognized early phase in host response that occurs prior to viral expression. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and sensitive cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

ecology↗