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Science (New York, N.Y.) Sep 2023CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies...
CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.
Topics: Animals; Humans; Mice; Cell Cycle Proteins; DNA Replication; DNA-Binding Proteins; Minichromosome Maintenance Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Protein Interaction Mapping; Computer Simulation; Nuclear Proteins; Dwarfism; Microcephaly; Xenopus laevis
PubMed: 37590370
DOI: 10.1126/science.adi3448 -
Cell Apr 2024Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts...
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Topics: Cell Nucleolus; Cell Nucleus; Nuclear Proteins; Proton-Motive Force; RNA; Phase Separation; Intrinsically Disordered Proteins; Animals; Xenopus laevis; Oocytes
PubMed: 38503281
DOI: 10.1016/j.cell.2024.02.029 -
Cell Feb 2024Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which...
Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.
Topics: Animals; Gene Expression; Myelin Sheath; Oligodendroglia; Retroelements; RNA; Zebrafish; Anura
PubMed: 38364788
DOI: 10.1016/j.cell.2024.01.011 -
Current Opinion in Neurobiology Oct 2023The transition from larval to adult locomotion in the anuran, Xenopus laevis, involves a dramatic switch from axial to appendicular swimming including intermediate... (Review)
Review
The transition from larval to adult locomotion in the anuran, Xenopus laevis, involves a dramatic switch from axial to appendicular swimming including intermediate stages when the tail and hindlimbs co-exist and contribute to propulsion. Hatchling tadpole swimming is generated by an axial central pattern generator (CPG) which matures rapidly during early larval life. During metamorphosis, the developing limbs are controlled by a de novo appendicular CPG driven initially by the axial system before segregating to allow both systems to operate together or independently. Neuromodulation plays important roles throughout, but key modulators switch their effects from early inhibitory influences to facilitating locomotion. Temperature affects the construction and operation of locomotor networks and global changes in environmental temperature place aquatic poikilotherms, like amphibians, at risk. The locomotor control strategy of anurans differs from other amphibian groups such as salamanders, where evolution has acted upon the thyroid hormone pathway to sculpt different developmental outcomes.
Topics: Animals; Spinal Cord; Larva; Locomotion; Swimming; Anura; Metamorphosis, Biological
PubMed: 37549591
DOI: 10.1016/j.conb.2023.102753 -
Nature Sep 2023Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells. Elucidation of how these effector proteins...
Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria. These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.
Topics: Animals; Bacterial Proteins; Cell Death; Fluorescein; Liposomes; Oocytes; Plant Cells; Plant Diseases; Porins; Protein Folding; Solutions; Water; Xenopus laevis; Osmolar Concentration
PubMed: 37704725
DOI: 10.1038/s41586-023-06531-5 -
Scientific Reports Aug 2023This article addresses two questions, why certain animals (frogs, breaststroke swimmers, hovering fliers, jellyfish) push rapidly against the surrounding fluid and then...
This article addresses two questions, why certain animals (frogs, breaststroke swimmers, hovering fliers, jellyfish) push rapidly against the surrounding fluid and then reach forward slowly, and whether this rhythm of propulsion is a manifestation of the universal phenomenon of design evolution in nature. Emphasis is on the distribution of time periods of locomotion in which, during the driving phase of cyclic movement (the motive stroke, phases 1 and 2, in alternating sequence with the dissipative stroke, phase 3), the work is generated (phase 1) and dissipated (phase 2). The relative lengths of the characteristic times t and t of the phases 1 and 2, are predicted. The relative duration of the proposed three phases of a cycle is the 'rhythm'. The analysis is based on a model of how the effective cross-sections of the stroking body parts impact the surrounding medium, water, or air, and the total power required to account for the kinetic energy losses during phases 2 and 3, which are due to drag forces posed by the surrounding medium. The body configuration (limbs' cross-sections) determines the limbs' velocities that maximize mean power, and the times t and t within the motive stroke. Emphasis is placed on the freedom to change the evolving design. Freedom is represented in two ways: the number of degrees of freedom in changing the dimensions of the model and its deformation in time, and the effect that evolutionary changes have on the access that the body has to its available space. Freedom to change the locomotion design leads to greater power and speed.
Topics: Animals; Locomotion; Anura; Cnidaria; Extremities; Fatigue
PubMed: 37640736
DOI: 10.1038/s41598-023-41023-6 -
International Journal of Molecular... Aug 2023Vasovagal syncope (VVS) refers to a heterogeneous group of conditions whereby the cardiovascular reflexes normally controlling the circulation are interrupted... (Review)
Review
Vasovagal syncope (VVS) refers to a heterogeneous group of conditions whereby the cardiovascular reflexes normally controlling the circulation are interrupted irregularly in response to a trigger, resulting in vasodilation, bradycardia, or both. VVS affects one-third of the population at least once in their lifetime or by the age of 60, reduces the quality of life, and may cause disability affecting certain routines. It poses a considerable economic burden on society, and, despite its prevalence, there is currently no proven pharmacological treatment for preventing VVS. The novel procedure of ganglionated plexus (GP) ablation has emerged rapidly in the past two decades, and has been proven successful in treating syncope. Several parameters influence the success rate of GP ablation, including specific ablation sites, localization and surgical techniques, method of access, and the integration of other interventions. This review aims to provide an overview of the existing literature on the physiological aspects and clinical effectiveness of GP ablation in the treatment of VVS. Specifically, we explore the association between GPs and VVS and examine the impact of GP ablation procedures as reported in human clinical trials. Our objective is to shed light on the therapeutic significance of GP ablation in eliminating VVS and restoring normal sinus rhythm, particularly among young adults affected by this condition.
Topics: Young Adult; Animals; Humans; Syncope, Vasovagal; Quality of Life; Ablation Techniques; Anura; Bradycardia
PubMed: 37686062
DOI: 10.3390/ijms241713264 -
Iranian Biomedical Journal Jul 2023The canonical Wnt signal transduction or the Wnt/β-catenin pathway plays a crucial role in both carcinogenesis and development of animals. Activation of the Gαq class...
BACKGROUND
The canonical Wnt signal transduction or the Wnt/β-catenin pathway plays a crucial role in both carcinogenesis and development of animals. Activation of the Gαq class of Gα proteins positively regulates Wnt/β-catenin pathway, and expression of Gαq in human embryonic kidney 293 (HEK293T) cells or Xenopus oocytes leads to the inhibition of glycogen synthase kinase-3 beta and cellular accumulation of β-catenin. This study investigated whether Gαq-mediated cellular accumulation of β-catenin could affect the transcriptional activity of this protein.
METHODS
HEK-293T and HT-29 cells were used for cell culture and transfection. Protein localization and quantification were assessed by using immunofluorescence microscopy, cell fractionation assay, and Western blotting analysis. Gene expression at the transcription level was examined by quantitative reverse transcriptase/real-time PCR method.
RESULTS
Transcription of two cellular β-catenin target genes (c-MYC and CCND1) and the β-catenin/ T-cell factor reporter luciferase gene (TopFlash plasmid) significantly increased by Gαq activation. The Gαq-mediated increase in the expression level of the β-catenin-target genes was sensitive to the expression of a minigene encoding a specific Gαq blocking peptide. The results of cell fractionation and Western blotting experiments showed that activation of Gαq signaling increased the intracellular β-catenin protein level, but it blocked its membrane localization.
CONCLUSION
Our results reveal that the Gαq-dependent cellular accumulation of β-catenin can enhance β-catenin transcriptional activity.
Topics: Animals; Humans; beta Catenin; Epithelial Cells; HEK293 Cells; Transcription, Genetic; Wnt Signaling Pathway; Xenopus
PubMed: 37481708
DOI: 10.61186/ibj.3890 -
Scientific Reports Oct 2023Exploring the timing of life-history transitions has been a pivotal focus in the field of evolutionary ecology. Studies on amphibian metamorphosis are well suited to...
Exploring the timing of life-history transitions has been a pivotal focus in the field of evolutionary ecology. Studies on amphibian metamorphosis are well suited to investigate this aspect. We propose a species-specific model to predict the optimal metamorphosis point for frog individuals with different larval growth trajectories. Because overall fitness will be determined throughout both aquatic and terrestrial stages, we included growth and survival rates of aquatic and terrestrial stages in the fitness equation. Then we conducted a rearing experiment on a brown frog, Rana ornativentris, as an example to obtain the size at metamorphosis, larval period, and larval growth trajectory. Based on these results, we determined the model's parameters to fit the actual metamorphosis patterns. Because the parameters are supposed to be evolutionarily maintained, our data-driven approach enabled obtaining fundamental ecological information (evolutionally-based life-history parameters) of the target species. Comparing the parameters among species will allow us to understand the mechanisms in determining life-history transition more deeply.
Topics: Humans; Animals; Metamorphosis, Biological; Anura; Larva; Ranidae; Morphogenesis
PubMed: 37783741
DOI: 10.1038/s41598-023-43639-0 -
Disease Models & Mechanisms May 2024Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic... (Review)
Review
Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic variants being identified demands in vivo model systems that are amenable to functional analyses at scale. Here we provide a practical guide for using the diploid frog species Xenopus tropicalis to study many genes and variants to uncover conserved mechanisms of pathobiology relevant to human disease. We discuss key considerations in modelling human genetic disorders: genetic architecture, conservation, phenotyping strategy and rigour, as well as more complex topics, such as penetrance, expressivity, sex differences and current challenges in the field. As the patient-driven gene discovery field expands significantly, the cost-effective, rapid and higher throughput nature of Xenopus make it an essential member of the model organism armamentarium for understanding gene function in development and in relation to disease.
Topics: Animals; Xenopus; Humans; Disease Models, Animal; Genetic Diseases, Inborn; Phenotype
PubMed: 38832520
DOI: 10.1242/dmm.050754