Changes in spindle morphology driven by TPX2 overexpression in MYC-driven breast cancer cells.

The MYC oncogene was previously shown to induce mitotic spindle defects, chromosome instability, and reliance on the microtubule-associated protein TPX2 to survive, but how TPX2 levels affect spindle morphology in cancer cells has not previously been examined in detail. We show that breast cancer cell lines expressing high levels of MYC and TPX2 possess shorter spindles with increased TPX2 localization at spindle poles. A similar effect was observed in non-transformed human RPE-1 cells compared to a tumor cell line (HeLa) that overexpresses MYC . These results demonstrate that TPX2 alters spindle length and morphology in cancer cells, which may contribute their ability to divide despite MYC-induced mitotic stress.

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies.

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

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, 123 KKLK 126 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.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran 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., arm-preserving) 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. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) 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 associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

A randomised controlled feasibility trial of an early years language development intervention: results of the 'outcomes of Talking Together evaluation and results' (oTTer) project.

BACKGROUND: Early language difficulties are associated with poor school readiness and can impact lifelong attainment. The quality of the early home language environment is linked to language outcomes. However, few home-based language interventions have sufficient evidence of effectiveness in improving preschool children's language abilities. This study reports the first stage in the evaluation of a theory-based programme, Talking Together (developed and delivered by BHT Early Education and Training) given over 6 weeks to families in the home setting. We aimed to test the feasibility and acceptability of delivering Talking Together in the Better Start Bradford community prior to a definitive trial, using a two-armed randomised controlled feasibility study. METHODS: Families from a single site within the Better Start Bradford reach area were randomly allocated (1:1) to the Talking Together intervention or a wait list control group. Child language and parent-level outcome measures were administered before randomisation (baseline), pre-intervention (pre-test), 2 months post-intervention start (post-test), and 6 months post-intervention start (follow-up). Routine monitoring data from families and practitioners were also collected for eligibility, consent, protocol adherence, and attrition rates. Descriptive statistics on the feasibility and reliability of potential outcome measures were analysed alongside qualitative feedback on trial design acceptability. Pre-defined progression-to-trial criteria using a traffic light system were assessed using routine monitoring data. RESULTS: Two-hundred and twenty-two families were assessed for eligibility; of these, 164 were eligible. A total of 102 families consented and were randomised (intervention: 52, waitlist control: 50); 68% of families completed outcome measures at 6-month follow-up. Recruitment (eligibility and consent) reached 'green' progression criteria; however, adherence reached 'amber' and attrition reached 'red' criteria. Child- and parent-level data were successfully measured, and the Oxford-CDI was identified as a suitable primary outcome measure for a definitive trial. Qualitative data not only indicated that the procedures were largely acceptable to practitioners and families but also identified areas for improvement in adherence and attrition rates. CONCLUSIONS: Referral rates indicate that Talking Together is a much-needed service and was positively received by the community. A full trial is feasible with adaptations to improve adherence and reduce attrition. TRIAL REGISTRATION: ISRCTN registry ISRCTN13251954. Retrospectively registered 21 February 2019.

Principles of genome activation in the early embryo.

A major hurdle in an embryo's life is the initiation of its own transcriptional program, a process termed Zygotic Genome Activation (ZGA). In many species, ZGA is intricately timed, with bulk transcription initiating at the end of a series of reductive cell divisions when cell cycle duration increases. At the same time, major changes in genome architecture give rise to chromatin states that are permissive to RNA polymerase II activity. Yet, we still do not understand the series of events that trigger gene expression at the right time and in the correct sequence. Here we discuss new discoveries that deepen our understanding of how zygotic genes are primed for transcription, and how these events are regulated by the cell cycle and nuclear import. Finally, we speculate on the evolutionary basis of ZGA timing as an exciting future direction for the field.

Mitotic chromosomes scale to nuclear-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 scale coordinately with mitotic spindles, but the 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 reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-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 α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

Polyploidy in Xenopus lowers metabolic rate by decreasing total cell surface area.

Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism 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. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na+/K+ ATPase, reducing 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.

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

Genome and cell size are strongly correlated across species1,2,3,4,5,6 and influence physiological traits like developmental rate.7,8,9,10,11,12 Although size scaling features such as the nuclear-cytoplasmic (N/C) ratio are precisely maintained in adult tissues,13 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 ancestral frog genome, ranging from 20 to 108 chromosomes.14,15 The most widely studied species, X. laevis (4N = 36) and X. tropicalis (2N = 20), scale at all levels, from body size to cellular and subcellular levels.16 Paradoxically, the rare, critically endangered dodecaploid (12N = 108) Xenopus longipes (X. longipes) is a small frog.15,17 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, whereas nuclear size correlated with genome size during embryogenesis, resulting in different N/C ratios in blastulae prior to gastrulation. 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.

Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time.

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

Molecular conflicts disrupting centromere maintenance 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 the 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 overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although 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 preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the 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.

Scaling of biosynthesis and metabolism with cell size.

Cells adopt a size that is optimal for their function, and pushing them beyond this limit can cause cell aging and death by senescence or reduce proliferative potential. However, by increasing their genome copy number (ploidy), cells can increase their size dramatically and homeostatically maintain physiological properties such as biosynthesis rate. Recent studies investigating the relationship between cell size and rates of biosynthesis and metabolism under normal, polyploid, and pathological conditions are revealing new insights into how cells attain the best function or fitness for their size by tuning processes including transcription, translation, and mitochondrial respiration. A new frontier is to connect single-cell scaling relationships with tissue and whole-organism physiology, which promises to reveal molecular and evolutionary principles underlying the astonishing diversity of size observed across the tree of life.

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 (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa 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 Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus 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.

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.

The power of amphibians to elucidate mechanisms of size control and scaling.

Size is a fundamental feature of biology that affects physiology at all levels, from the organism to organs and tissues to cells and subcellular structures. How size is determined at these different levels, and how biological structures scale to fit together and function properly are important open questions. Historically, amphibian systems have been extremely valuable to describe scaling phenomena, as they occupy some of the extremes in biological size and are amenable to manipulations that alter genome and cell size. More recently, the application of biochemical, biophysical, and embryological techniques to amphibians has provided insight into the molecular mechanisms underlying scaling of subcellular structures to cell size, as well as how perturbation of normal size scaling impacts other aspects of cell and organism physiology.

Emergent properties of mitotic chromosomes.

As a cell prepares to divide, its genetic material changes dramatically in both form and function. During interphase, a dynamic interplay between DNA compartmentalization and transcription functions to program cell identity. During mitosis, this purpose is put on hold and instead chromosomes function to facilitate their accurate segregation to daughter cells. Chromatin loops are rearranged, stacked, and compressed to form X-shaped chromosomes that are neatly aligned at the center of the mitotic spindle and ready to withstand the forces of anaphase. Many factors that contribute to mitotic chromosome assembly have now been identified, but how the plethora of molecular mechanisms operate in concert to give rise to the distinct form and physical properties of mitotic chromosomes at the cellular scale remains under active investigation. In this review, we discuss recent work that addresses a major challenge for the field: How to connect the molecular-level activities to large-scale changes in whole-chromosome architecture that determine mitotic chromosome size, shape, and function.

A randomised controlled feasibility trial and qualitative evaluation of an early years language development intervention: study protocol of the 'outcomes of Talking Together evaluation and results' (oTTer) project.

BACKGROUND: Problems with oral language skills in childhood have been linked with poor educational, employment, and mental health outcomes. In the UK, there is increasing concern about the oral language skills of children, particularly children from areas of social disadvantage. Research emphasises the importance of the home language environment as a fundamental bedrock for the development of oral language skills. It is vital, therefore, that support is available to help families in need to provide the optimal language environment for their child. Talking Together is a 6-week home visiting programme recently commissioned by Better Start Bradford to develop parents' knowledge of the importance of a good language environment and help to improve parent-child interactions. This study represents the initial steps in developing a definitive trial of the Talking Together programme. METHOD: This study is a two-arm randomised controlled feasibility study in which families referred into the Talking Together programme and consent to participate in the trial will be randomly allocated to either an intervention group or a waiting control group. We will assess the recruitment and retention rates, the representativeness of our sample, the appropriateness of our measures, and the sample size needed for a definitive trial. We will also carry out a qualitative evaluation to explore the acceptability of trial procedures for families and service providers, fidelity of delivery, time and resources for training, and barriers and facilitators to engagement with the programme. Clear progression criteria will be used to assess suitability for a definitive trial. CONCLUSION: This feasibility study will inform the development of a definitive trial of this home-based visiting programme, which will add to the sparse evidence base on which practitioners can draw when supporting families in need. The lessons learnt from this feasibility study will also inform the wider evaluation work of the Better Start Bradford Innovation Hub. TRIAL REGISTRATION: The trial is registered with the ISRCTN registry: study ID ISRCTN13251954. Date of registration: 21 February 2019 (the trial was retrospectively registered).

Kif2a Scales Meiotic Spindle Size in Hymenochirus boettgeri.

Size is a fundamental feature of biological systems that affects physiology at all levels. For example, the dynamic, microtubule-based spindle that mediates chromosome segregation scales to a wide range of cell sizes across different organisms and cell types. Xenopus frog species possess a variety of egg and meiotic spindle sizes, and differences in activities or levels of microtubule-associated proteins in the egg cytoplasm between Xenopus laevis and Xenopus tropicalis have been shown to account for spindle scaling [1]. Increased activity of the microtubule severing protein katanin scales the X. tropicalis spindle smaller compared to X. laevis [2], as do elevated levels of TPX2, a protein that enriches the cross-linking kinesin-5 motor Eg5 at spindle poles [3]. To examine the conservation of spindle scaling mechanisms more broadly across frog species, we have utilized the tiny, distantly related Pipid frog Hymenochirus boettgeri. We find that egg extracts from H. boettgeri form meiotic spindles similar in size to X. tropicalis but that TPX2 and katanin-mediated scaling is not conserved. Instead, the microtubule depolymerizing motor protein kif2a functions to modulate spindle size. H. boettgeri kif2a possesses an activating phosphorylation site that is absent from X. laevis. Comparison of katanin and kif2a phosphorylation sites across a variety of species revealed strong evolutionary conservation, with X. laevis and X. tropicalis possessing distinct and unique alterations. Our study highlights the diversity and complexity of spindle assembly and scaling mechanisms, indicating that there is more than one way to assemble a spindle of a particular size.

Cell Biology: The Health Hazards of Super-Sizing.

Cells typically occupy a narrow range of sizes according to their type. A new study reveals that cells grown to gigantic proportions fail to synthesize sufficient macromolecules, resulting in cytoplasm dilution and a loss of fitness reminiscent of old cells.

Generation of Xenopus Haploid, Triploid, and Hybrid Embryos.

Frog species of the genus Xenopus are widely used for studies of cell and developmental biology, and recent genome sequencing has revealed interesting phylogenetic relationships. Here we describe methods to generate haploid, triploid, and hybrid species starting from eggs and sperm of Xenopus laevis and Xenopus tropicalis that enable investigation of how genome size and content affect physiology at the organismal, cellular, and subcellular levels.