PARP1-dependent DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.

Characterization of a vacuolar importer of secologanin in Catharanthus roseus.

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drug modulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium may alter this balance and modulate the ability of PNU to potentiate α7.

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Mechanical characterization of Xenopus laevis oocytes using atomic force microscopy.

Mechanical properties are essential for the biological activities of cells, and they have been shown to be affected by diseases. Therefore, accurate mechanical characterization is important for studying the cell lifecycle, cell-cell interactions, and disease diagnosis. While the cytoskeleton and actin cortex are typically the primary structural stiffness contributors in most live cells, oocytes possess an additional extracellular layer known as the vitelline membrane (VM), or envelope, which can significantly impact their overall mechanical properties. In this study, we utilized nanoindentation via an atomic force microscope to measure the Young's modulus of Xenopus laevis oocytes at different force setpoints and explored the influence of the VM by conducting measurements on oocytes with the membrane removed. The findings revealed that the removal of VM led to a significant decrease in the apparent Young's modulus of the oocytes, highlighting the pivotal role of the VM as the main structural component responsible for the oocyte's shape and stiffness. Furthermore, the mechanical behavior of VM was investigated through finite element (FE) simulations of the nanoindentation process. FE simulations with the VM Young's modulus in the range 20-60 MPa resulted in force-displacement curves that closely resemble experimental in terms of shape and maximum force for a given indentation depth.

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.

Robust trigger wave speed in Xenopus cytoplasmic extracts.

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we find that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derive a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

The people behind the papers - Paul Huber and Carole LaBonne.

The bromodomain and extra-terminal (BET) family of proteins reads epigenetic histone acetylation marks on the genome and regulates the transcriptional machinery. In their study, Carole LaBonne and colleagues reveal the role of BET protein activity in the maintenance of pluripotency and establishment of the neural crest in Xenopus laevis. To know more about their work, we spoke to the first author Paul Huber and the corresponding author Carole LaBonne, Developmental and Stem Cell Biologist at Northwestern University.

Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Sox21 homeologs autoregulate expression levels to control progression through neurogenesis.

The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeologs of sox21, namely sox21.S and sox21.L. Previous studies focused on Sox21.S, but its amino acid sequence is divergent, lacking conserved poly-A stretches and bearing more similarity with ancestral homologs. In contrast, Sox21.L shares higher sequence similarity with mouse and chick Sox21. To determine if Sox21.S and Sox21.L have distinct functions, we conducted gain and loss-of-function studies in Xenopus embryos. Our studies revealed that Sox21.S and Sox21.L are functionally redundant, but Sox21.L is more effective at driving changes than Sox21.S. These results also support our earlier findings in ectodermal explants, demonstrating that Sox21 function is dose-dependent. While Sox21 is necessary for primary neuron formation, high levels prevent their formation. Strikingly, these proteins autoregulate, with high levels of Sox21.L reducing sox21.S and sox21.L mRNA levels, and decreased Sox21.S promoting increased expression of sox21.L. Our findings shed light on the intricate concentration-dependent roles of Sox21 homeologs in Xenopus neurogenesis.

Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor.

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

Unravelling nicotinic receptor and ligand features underlying neonicotinoid knockdown actions on the malaria vector mosquito Anopheles gambiae.

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.

Targeting pericentric non-consecutive motifs for heterochromatin initiation.

Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1-3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.

Expression analysis of thg1l during Xenopus laevis development.

The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.

Amphibian mast cells serve as barriers to chytrid fungus infections.

Global amphibian declines are compounded by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Much has been learned about the roles of amphibian skin-produced antimicrobial components and microbiomes in controlling Bd, yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. This includes a significant reduction in infiltration of Bd-infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventing Bd-mediated changes to skin microbiomes. Mammalian mast cells are known for their production of the pleiotropic interleukin-4 (IL4) cytokine and our findings suggest that the X. laevis IL4 plays a key role in manifesting the effects seen following cutaneous mast cell enrichment. Together, this work underscores the importance of amphibian skin-resident immune cells in anti-Bd defenses and illuminates a novel avenue for investigating amphibian host-chytrid pathogen interactions.

Zbtb11 interacts with Otx2 and patterns the anterior neuroectoderm in Xenopus.

The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.

Dynamic BTB-domain filaments promote clustering of ZBTB proteins.

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine.

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling researchers to model human disease. Recording and tracking individual animals allows researchers to reduce the number of animals required for study and refine practices to improve animal wellbeing. Several well-documented methods exist for marking and tracking mammals, including ear punching and ear tags. However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly. In this manuscript, we outline methods and best practices for marking Xenopus laevis with a rotary tattoo machine. Proper tattooing results in high-quality tattoos, making individuals easily distinguishable for researchers and posing minimal risk to animals' health. We also highlight the causes of poor-quality tattoos, which can result in tattoos that fade quickly and cause unnecessary harm to animals. This approach allows researchers and veterinarians to mark amphibians, enabling them to track biological replicates and transgenic lines and to keep accurate records of animal health.

Unveiling vertebrate development dynamics in frog Xenopus laevis using micro-CT imaging.

BACKGROUND: Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism in various biological disciplines, prominently in developmental biology to study body plan reorganization during metamorphosis. However, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus' late developmental stages. FINDINGS: This study utilized micro-computed tomography (micro-CT), a noninvasive 3-dimensional (3D) imaging technique with micrometer-scale resolution, to explore the developmental dynamics and morphological changes in Xenopus laevis. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adults. This dataset enhances our understanding of vertebrate development and supports various analyses. We conducted a careful examination, analyzing body size, shape, and morphological features, focusing on skeletogenesis, teeth, and organs like the brain and gut at different stages. Our analysis yielded valuable insights into 3D morphological changes during Xenopus' development, documenting details previously unrecorded. These datasets hold the solid potential for further morphological and morphometric analyses, including segmentation of hard and soft tissues. CONCLUSIONS: Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus' development and the associated morphological changes in the future. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans can be used in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.