Biology subjects

Lipinszki, Z.

Publications and source records attributed to Lipinszki, Z..

11 recordsLinked to original sources

bioRxiv2026

Functionally dark genes and the transcriptomic landscape of sporulation in a model mushroom-forming fungus

Spores are the primary means of fungal reproduction, contributing to genetic diversity, colonization, and adaptation. Although spore formation is a pivotal part of the fungal life cycle, its genetic underpinnings remain poorly known. In this study, we characterize transcriptomic changes from late meiosis to early basidiospore formation in the mushroom-forming fungus Coprinopsis cinerea, decipher several cellular processes, and identify novel genes involved in this process. We identify distinct trajectories of gene expression, each of which display different functional signals, corresponding to meiotic and morphogenetic processes and transitions between these. Our analyses identify diverse arrays of fungal cell wall modifying carbohydrate-active enzymes, ferritins, a putative catechol-melanin synthesis pathway, as well as components of the mitotic/meiotic apparatus. We present twelve highly conserved genes with roles specific to sexual sporulation in both budding yeast and C. cinerea, indicating deep conservation of the gene networks driving sexual spore formation. Reverse genetics identified three conserved but functionally poorly characterized genes conferring sporeless and spore-poor phenotypes that result from postmeiotic developmental arrests stemming from spore inflation and nuclear migration problems. Overall, this study provides novel insight into basidiomycete spore formation and highlights the cornucopia of novel functions encoded by functionally dark genes.

microbiology↗

bioRxiv2026

Coordinated regulation of Barrier-to-Autointegration Factor (BAF) phosphorylation by protein phosphatases PP2A and PP4

Phosphorylation is crucial for the regulation of Barrier-to-Autointegration Factor (BAF), a conserved nuclear envelope (NE)-associated protein that, among its diverse functions, binds chromatin and mediates its anchoring to the NE. In interphase, cycles of BAF phosphorylation, which inhibits chromatin binding, and dephosphorylation, which restores it, are essential for the dynamic anchoring of chromatin to the NE. During mitosis, BAF becomes highly phosphorylated and dissociates from chromatin, except for a small centromeric pool (cenBAF). Upon mitotic exit, BAF is dephosphorylated and re-associates with chromosomes. Two Ser/Thr protein phosphatases, PP2A and PP4, are known to dephosphorylate BAF, but their specific roles in regulating BAF phosphorylation are not yet fully understood. Here, we show that PP2A is the main phosphatase driving BAF dephosphorylation in interphase, whereas PP4 plays a minor role. During mitosis, both PP2A and PP4 localize to centromeres; however, only PP4 directly interacts with cenBAF, suggesting that it maintains this fraction in an unphosphorylated state. Notably, PP4s centromeric localization depends on PP2A. At mitotic exit, PP2A dephosphorylates the large pool of mitotically phosphorylated BAF, while PP4 controls the timing of its re-association with chromatin. Together, these findings reveal a coordinated interplay between PP2A and PP4 in controlling BAF dephosphorylation and function.

Molecular Biology↗

bioRxiv2026

Intrahepatic reporter assay reveals leaky somatic blockade of L1 retrotransposition in mice

Long interspersed element-1 (LINE-1, L1) retrotransposition has long been proposed to occur in somatic tissues, yet direct experimental evidence distinguishing adult somatic events from early embryonic insertions has remained limited. Here we establish an intrahepatic L1 reporter assay that enables immunohistochemical detection and quantitative analysis of L1 retrotransposition in vivo. Using autonomous and non-autonomous L1 reporter variants, we demonstrate clearly detectable somatic L1 activity in the mouse liver. Comparative analysis of L1 activity in liver tissue and tumor-derived cell culture reveals that tumor cells preferentially restrict L1 at early regulatory stages, consistent with epigenetic control, whereas downstream defence mechanisms are comparatively permissive. In contrast, normal liver tissue shows stronger restriction at later stages of the L1 life cycle. Together, our results provide direct experimental evidence for somatic L1 retrotransposition in vivo in adult liver and reveal distinct regulatory strategies that shape L1 activity in tumor versus normal somatic cells. TeaserGenome destabilizing L1 retrotransposon activity is present in somatic tissues, where it likely contributes to cancer development.

genetics↗

bioRxiv2025

Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but their role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species genome. Transcriptome analyses identified several misregulated genes in the {Delta}gcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.

microbiology↗

bioRxiv2024

Evidencing the role of a conserved polar signaling channel in the activation mechanism of the u-opioid receptor

The activity of G protein-coupled receptors has been generally linked to dynamically interconverting structural and functional states and the process of activation was proposed to be controlled by an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extent of functional effect exert their actions. According to our recent hypothesis, the transmission of the external stimulus is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of conserved polar amino acids and the rearrangement of polar species. Previously, we have examined the -opioid, {beta}2-adrenergic and type 1 cannabinoid receptors by performing molecular dynamics simulations. Results revealed correlated dynamics of a polar signaling channel connecting the orthosteric binding pocket and the intracellular G protein-binding surface in all three class A receptors. In the present study, the interplay of this polar signaling channel in the activation mechanism was evidenced by systematic mutation of the channel residues of the -opioid receptor. Mutant receptors were analyzed utilizing molecular dynamics simulations and characterized in vitro by means of radioligand receptor binding and G protein stimulation assays. Apart from one exception, all mutants failed to bind the endogenous agonist endomorphin-2 and to stimulate the Gi protein complex. Furthermore, mutation results confirmed strong allosteric coupling between the binding pocket and the intracellular surface. The strong association and optimal bioactive orientation of the bound agonist was found to be crucial for the initiation of correlated motions and consequent signaling.

molecular biology↗

bioRxiv2024

A new regulator of sporulation sheds light on spore morphogenesis and ballistospory in mushroom-forming fungi

Sporulation is the most widespread means of reproduction and dispersal in fungi. In the Basidiomycota, sexual spores are produced on specialised cells known as basidia, from which they are discharged forcibly by a powered process called ballistospory, the highest known acceleration in nature. However, the genetics of sporulation, in particular postmeiotic events related to spore morphogenesis and ballistospory, remain poorly known. Here, we explore the genetics of these processes, based on a new, highly conserved transcription factor, Sporulation-Related Regulator 1 (SRR1), and its putative downstream regulatory network. Reverse genetics of Srr1 in the model mushroom Coprinopsis cinerea and commercially produced oyster mushroom indicated a conserved role of Srr1 in sporulation across Agaricomycetes. RNA-Seq analysis and motif-based inference of a hypothetical SRR1 gene regulatory network allowed delimiting putative targets regulated by SRR1 in a direct and indirect manner. Using this network and comparative genomics, we identified genes associated with ballistospory, including a putative SRR1-target chitinase, which was found to be required for normal spore production and morphology. Overall, our study offers new insights into the genetic mechanisms governing postmeiotic spore morphogenesis and ballistospory in the Agaricomycetes.

microbiology↗

bioRxiv2024

Retracing the horizontal transfer of a novel innate immune factor in Drosophila

Immune systems are among the most dynamically evolving traits across the tree of life, and long-lived macroparasites play an outsized role in shaping animal immunity. Even without adaptive immunity, insects have evolved potent innate immune strategies to neutralize such enemies, including nematodes and parasitoid wasps. One such strategy relies on endosymbioses between insects and toxin-expressing bacteria. Here, we use genome editing in Drosophila melanogaster to retrace the evolution of two of such toxins -- cytolethal distending toxin B (cdtB) and apoptosis inducing protein of 56kDa (aip56) -- that were horizontally transferred from bacteriophages to insects. We found that a cdtB::aip56 fusion gene (fusionB), which is conserved in Drosophila ananassae subgroup species, dramatically promoted fly survival and suppressed wasp development when expressed in D. melanogaster immune tissues. FusionB, a functional nuclease, was secreted into the host hemolymph where it targeted the parasitoid embryos serosal tissue and is to our knowledge the first humoral anti-parasitoid toxin in Drosophila. When expressed ubiquitously, fusionB slowed development in late stage fly larvae and eventually killed flies, pointing to the salience of regulatory constraint in preventing autoimmunity. Our findings demonstrate how horizontal gene transfer, in the right regulatory context, can instantly provide new and potent innate immune modules in animals.

evolutionary biology↗

bioRxiv2024

Distinct components of nucleoside-modified messenger RNA vaccines cooperate to instruct efficient germinal center responses

Nucleoside-modified mRNA vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cells. The lipid nanoparticle (LNP) component of mRNA vaccines possesses inherent adjuvant activity. However, to what extent the nucleoside-modified mRNA can be sensed and contribute to Tfh cell responses remains largely undefined. Herein, we deconvoluted the signals induced by LNP and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrated that the nucleoside-modified mRNA drives the production of type I interferons that act on DCs to induce their maturation and the induction of Th1-biased Tfh responses. Conversely, LNP favors the acquisition of a Tfh cell-inducing program in DCs, a stronger Th2 polarization in Tfh cells, and allows for rapid mRNA translation by DCs within the draining lymph node. Our work unravels distinct adjuvant features of mRNA and LNP necessary for the induction of Tfh cells, with implications for vaccine design.

immunology↗

bioRxiv2024

Gamma-TuRC proteins contribute to the dynamic organization of MTOCs during Drosophila spermatogenesis

The initiation of microtubule formation is facilitated by {gamma}-tubulin and {gamma}-Tubulin Ring Complex ({gamma}-TuRC) in various microtubule-organizing centers (MTOCs). While the heterogeneity of tissue-specific MTOCs and {gamma}-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. We investigated the testis-specific distribution and biochemical interaction of the canonical {gamma}-TuRC proteins Grip163 and Grip84. We found that while Grip163 is present on the centrosome and basal body, Grip84 localizes to the centrosome and Golgi in spermatocytes and colocalizes with the testis-specific {gamma}-TuRC at the basal body, apical nuclear tip, and near the elongated mitochondria after meiosis. We also show the apical nuclear tip localization of some {gamma}-TuRC interacting partners and prove their binding to testis-specific {gamma}-TuRC proteins. These results highlight and prove the importance of the different {gamma}-TuRCs in organizing the diverse MTOCs present during the extensive rearrangement of cell organelles during the spermatogenesis of Drosophila.

cell biology↗

bioRxiv2023

Protein phosphatase 4 is required for Centrobin function in DNA damage repair

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which result in similarly abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms and interrelationships of PP4 and Ctb in the regulation of HR repair.

cell biology↗

bioRxiv2018

Enhancing the translational capacity of E. coli by resolving the codon bias

Escherichia coli is a well-established, and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation.\n\nHere we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a ribosomal RNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in exponential growth phase.\n\nResults from expression assays of a panel of heterologous proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.\n\nImportanceCodon composition not fitting the codon bias of the expression host frequently compromises the efficient production of foreign proteins in E. coli. Various attempts to remedy the problem (codon optimization by gene synthesis, expression of rare tRNAs from a plasmid) proved to be unsatisfying. Our new approach, adjusting the tRNA pool by co-expressing extra copies of rare tRNA genes with ribosomal RNA genes, does not affect normal cell physiology, and seems to be a superior solution in terms of simplicity, cost, and yield.

synthetic biology↗