Biology subjects

Heald, R.

Publications and source records attributed to Heald, R..

At least 19 recordsLinked to original sources

bioRxiv2026

TOPBP1-Dependent DNA Damage Processing Promotes Centromere Loss in an Inviable Xenopus Hybrid

Hybrid incompatibility is associated with chromosome instability, DNA damage, and embryonic lethality, yet how cells respond to genome instability in hybrids is unclear. In inviable hybrids generated by fertilizing Xenopus tropicalis eggs with Xenopus laevis sperm, CENP-A is lost from paternal chromosomes, leading to elimination of two chromosome arms. Using hybrid Xenopus egg extract reactions, we show that CENP-A is removed from X. laevis chromosomes exposed to X. tropicalis cytoplasm through an RNA polymerase II-dependent process associated with DNA damage. Topoisomerase II Binding Protein 1 (TOPBP1) localized to damaged acentric chromosomes during metaphase, and its depletion impaired chromosome alignment, increased DNA damage, and blocked CENP-A removal. Inhibition of DNA polymerase theta increased DNA damage while promoting CENP-A retention, indicating that DNA damage processing rather than damage itself promotes CENP-A loss. Together, our findings reveal separable roles for TOPBP1 in chromosome organization and centromere destabilization, showing how a protective genome surveillance pathway can instead drive chromosome instability in an inviable hybrid.

cell biology↗

bioRxiv2026

Spindle scaling promotes adaptation to polyploidy

How the cell division machinery adapts to increases in genome copy number (ploidy) remains an open question with implications for evolution and cancer. Using Xenopus egg extracts and species spanning 2N to 12N ploidy, we found that stepwise increases in ploidy drove spindle multipolarity, accompanied by larger spindles, increased microtubule density, and differential scaling of spindle assembly factor localization. In contrast, extracts from the dodecaploid species Xenopus longipes supported robust bipolar spindle assembly at all ploidies. Engineering X. laevis extracts to mimic X. longipes by modulating microtubule regulators frequently overexpressed in cancer, or confining spindles inside small droplets, decreased spindle length and significantly rescued bipolarity. Thus, tuning spindle microtubule dynamics and architecture enables adaptation to increased chromosome number across acute and evolutionary timescales.

cell biology↗

bioRxiv2025

Ploidy and neuron size impact nervous system development and function in Xenopus

Neuron size varies significantly over evolution, contributing to diverse nervous systems of variable complexity, while aberrant neuron size is associated with neurodevelopmental and degenerative diseases. How do neuron cell body and neurite size and organization impact nervous system development and function? To systematically study effects of neuron size on the vertebrate nervous system, we characterized triploid Xenopus tadpoles that possess a 1.5-fold increase in genome size compared to diploids. Triploids displayed a linear increase in total neuronal volume and a superlinear increase in membrane surface area. Imaging, flow cytometry, and RNA-seq analyses revealed that triploid brains were morphologically and transcriptionally similar to diploid brains, but less proliferative, containing fewer neurons and displaying increased global activity. Interestingly, physiological differences at the neuron and nervous system levels affected swimming behavior in tadpoles. Our findings thus establish a framework to link genome size, neuron size, and nervous system development and function in vertebrates.

cell biology↗

bioRxiv2024

Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts

The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we investigate the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibited a broader range of bundle sizes, while X. tropicalis spindles contained mostly smaller bundles. Moreover, while both species exhibited larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise consisted of very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring underlying mechanisms. SIGNIFICANCE STATEMENTO_LICorrect spindle morphology is key to its function; however, traditional microscopy methods limit our view of spindle architecture. This study addresses the gap in resolving detailed spindle microtubule organization by using advanced imaging. C_LIO_LIThe research utilizes Expansion Microscopy (ExM) to reveal previously unobservable details of spindle morphology in egg extracts of two Xenopus species (X. laevis and X. tropicalis). This approach provides unprecedented clarity on microtubule arrangement and variations in spindle architecture. C_LIO_LIThis work establishes a new protocol for high-resolution imaging of spindle structures, offering insights into how spindle architecture is adapted in differently-sized spindles to ensure proper function. C_LI

cell biology↗

bioRxiv2024

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused [~]50% slower, while diffusion of positively charged proteins was reduced by [~]80-90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, negatively charged proteins of different sizes showed similar diffusivity suppression in extract, which are typically prepared under conditions that inhibit actin polymerization. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks. Significance StatementThe complex intracellular molecular environment is notably challenging to elucidate and recapitulate. Xenopus egg extracts provide a native yet manipulatable cytoplasm model. Through single-molecule microscopy, here we decipher the cytoplasmic environment and molecular interactions by examining the diffusion patterns of diverse proteins in Xenopus egg extracts with strategic manipulations. These experiments reveal an overwhelmingly negatively charged macromolecular environment with crosslinked meshworks, offering new insight into the inner workings of the cell.

biophysics↗

bioRxiv2024

The nuclear-cytoplasmic ratio controls the cell cycle period in compartmentalized frog egg extract

Each proliferating cell replicates its DNA and internal components before distributing this material evenly to its daughters. Although the regulation of cyclin-dependent kinases (Cdks) that dictate orderly cell cycle progression is well characterized, how the subcellular localization of the cell cycle machinery contributes to timing is not well understood. We investigated the influence of the nucleus by reconstituting cell cycle oscillations in droplets of frog egg extract in the absence or presence of a nuclear compartment and monitoring dynamics by time-lapse microscopy. We found that the cell cycle time increased in the presence of nuclei, which grew larger with each cell cycle. The correlation between increasing nuclear volume and a longer cell cycle period was maintained across extracts and nuclei from various Xenopus species and persisted upon inhibition of DNA replication or transcription. However, inhibition of nuclear import or the kinase Wee1 impacted the relationship between the nuclear-cytoplasmic ratio and the cell cycle period. To conceptually capture these experimental observations, we developed a computational model that incorporates cell cycle oscillations, nuclear-cytoplasmic compartmentalization, and periodic nuclear envelope breakdown and reformation. Altogether, our results support the major role of the nuclear compartment in setting the pace of the cell cycle and provide an explanation for the increase in cell cycle length observed at the midblastula transition when cells become smaller and the nuclear-cytoplasmic ratio increases.

biophysics↗

bioRxiv2024

Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway

The transition from meiotic divisions in the oocyte to embryonic mitoses is a critical step in animal development. Despite negligible changes to cell size and shape, following fertilization the small, barrel-shaped meiotic spindle is replaced by a large zygotic spindle that nucleates abundant astral microtubules at spindle poles. To probe underlying mechanisms, we applied a drug screening approach using Ciona eggs and found that inhibition of Casein Kinase 2 (CK2) caused a shift from meiotic to mitotic-like spindle morphology with nucleation of robust astral microtubules, an effect reproduced in cytoplasmic extracts prepared from Xenopus eggs. In both species, CK2 activity decreased at fertilization. Phosphoproteomic differences between Xenopus meiotic and mitotic extracts that also accompanied CK2 inhibition pointed to RanGTP-regulated factors as potential targets. Interfering with RanGTP-driven microtubule formation suppressed astral microtubule growth caused by CK2 inhibition. These data support a model in which CK2 activity attenuation at fertilization leads to activation of RanGTP-regulated microtubule effectors that induce mitotic spindle morphology.

cell biology↗

bioRxiv2024

Identification of a motif in TPX2 that regulates spindle architecture in Xenopus egg extracts

A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and architecture varies dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we identify a conserved nuclear localization sequence (NLS) motif, 123KKLK126 in X. laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA dramatically increased spontaneous formation of microtubule asters and recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the recruitment and activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.

cell biology↗

bioRxiv2022

Mitotic chromosomes scale to nucleo-cytoplasmic ratio and cell size in Xenopus

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to coordinately scale with mitotic spindles, but underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be re-set by cytoplasmic factors from earlier developmental stages. In vitro, increasing nucleo-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin scales mitotic chromosomes to cell surface area/volume (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and analysis of Hi-C data suggest that mitotic chromosomes scale through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

cell biology↗

bioRxiv2022

Ploidy modulates cell size and metabolic rate in Xenopus embryos

A positive correlation between genome size and cell size is well documented, but impacts on animal physiology are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Treatments that altered cell membrane stability or electrical potential abolished this difference, suggesting that a decrease in total cell surface area reduces basal energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism. One-Sentence SummaryThe amount of DNA per cell in a vertebrate modulates basal metabolism by altering cell size and plasma membrane energetics.

cell biology↗

bioRxiv2022

Small-molecule Polθ inhibitors provide safe and effective tumor radiosensitization in preclinical models

DNA polymerase theta (Pol{theta}) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Pol{theta} has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Pol{theta} DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, normal fibroblasts are not radiosensitized by Pol{theta} inhibition. Mechanistically, we show that the radiosensitization caused by Pol{theta} inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Pol{theta} inhibitor with improved metabolic stability. In vivo, the combination of Pol{theta} inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared to radiation alone. These results pave the way for future clinical trials of Pol{theta} inhibitors in combination with radiotherapy.

cancer biology↗

bioRxiv2022

Dodecaploid Xenopus longipes provides insight into the emergence of size scaling relationships during development

Genome and cell size are strongly correlated across species1-6 and influence physiological traits like developmental rate7-12. Although size scaling features such as the nuclear-cytoplasmic (N/C) ratio are precisely maintained in adult tissues13, it is unclear when during embryonic development size scaling relationships are established. Frogs of the genus Xenopus provide a model to investigate this question, since 29 extant Xenopus species vary in ploidy from 2 to 12 copies (n) of the genome, ranging from 20 to 108 chromosomes14,15. The most widely studied species, X. laevis (4n=36) and X. tropicalis (2n=20), scale at all levels from body size to cellular and subcellular16. Paradoxically, the rare, critically endangered dodecaploid (2n=108) X. longipes is a small frog15,17. We observed that despite some morphological differences, X. longipes and X. laevis embryogenesis occurred with similar timing, with genome to cell size scaling emerging at the swimming tadpole stage. Across the three species, cell size was determined primarily by egg size, while nuclear size correlated with genome size during embryogenesis, resulting in different nuclear-cytoplasmic ratios at the mid-blastula transition. At the subcellular level, nuclear size correlated more strongly with genome size, whereas mitotic spindle size scaled with cell size. Our cross-species study indicates that scaling of cell size to ploidy is not due to abrupt changes in cell division timing, that different size scaling regimes occur during embryogenesis, and that the developmental program of Xenopus is remarkably consistent across a wide range of genome and egg sizes. ONE SENTENCE SUMMARYComparison of size metrics in embryos from different ploidy Xenopus species, including the dodecaploid X. longipes, reveals distinct size scaling regimes during development.

developmental biology↗

bioRxiv2022

Molecular conflicts disrupting centromere assembly contribute to Xenopus hybrid inviability

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or over-expressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, whereas the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not, and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA Polymerase I or by preventing collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids. ONE SENTENCE SUMMARYCentromere incompatibilities in inviable Xenopus hybrids are sequence-independent and result from disruption of epigenetic pathways required for centromere maintenance.

cell biology↗

bioRxiv2022

Reconstitution of muscle cell microtubule organization in vitro

Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.

cell biology↗

bioRxiv2021

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs

Frogs are an ecologically diverse and phylogenetically ancient group of living amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (HiC) data for all species. Abundant satellite repeats occupy the unusually long ([~]20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl configuration similar to the "bouquet" structure of meiotic cells. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

genomics↗

bioRxiv2020

Identification and characterization of centromeric sequences in Xenopus laevis

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

genomics↗

bioRxiv2018

Xenopus hybrids provide insight into cell and organism size control

Determining how size is controlled is a fundamental question in biology that is poorly understood at the organismal, cellular and subcellular levels. The Xenopus species, X. laevis and X. tropicalis differ in size at all three of these levels. Despite these differences, fertilization of X. laevis eggs with X. tropicalis sperm gives rise to viable hybrid animals that are intermediate in size. We observed that although hybrid and X. laevis embryogenesis initiates from the same sized zygote and proceeds synchronously through development, hybrid animals were smaller by the tailbud stage, and a change in the ratio of nuclear size to cell size was observed shortly after zygotic genome activation (ZGA), suggesting that differential gene expression contributes to size differences. Transcriptome analysis at the onset of ZGA identified twelve transcription factors paternally expressed in hybrids. A screen of these X. tropicalis factors by expression in X. laevis embryos revealed that Hes7 and Ventx2 significantly reduced X. laevis body length size by the tailbud stage, although nuclear to cell size scaling relationships were not affected as in the hybrid. Together, these results suggest that transcriptional regulation contributes to biological size control in Xenopus.

developmental biology↗

bioRxiv2018

Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

cell biology↗