Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling.

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.

Microtubules: 50 years on from the discovery of tubulin.

Next year will be the 50th anniversary of the discovery of tubulin. To celebrate this discovery, six leaders in the field of microtubule research reflect on key findings and technological breakthroughs over the past five decades, discuss implications for therapeutic applications and provide their thoughts on what questions need to be addressed in the near future.

Katanin contributes to interspecies spindle length scaling in Xenopus.

Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure.

Mechanisms of intracellular scaling.

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, which places variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components scale to accommodate cell size differences. Nuclear transport has emerged as a conserved means of scaling nuclear size. A meiotic spindle scaling factor has been identified as the microtubule-severing protein katanin, which is differentially regulated by phosphorylation in two different-sized frog species. Anaphase mechanisms and levels of chromatin compaction both act to coordinate cell size with spindle and chromosome dimensions to ensure accurate genome distribution during cell division. Scaling relationships and mechanisms for many membrane-bound compartments remain largely unknown and are complicated by their heterogeneity and dynamic nature. This review summarizes cell and organelle size relationships and the experimental approaches that have elucidated mechanisms of intracellular scaling.

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.

Nuclear size is regulated by importin α and Ntf2 in Xenopus.

The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.

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.

Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus.

Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here we apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

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.

Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos.

Microtubule remodeling is critical for cellular and developmental processes underlying morphogenetic changes and for the formation of many subcellular structures. Katanins are conserved microtubule severing enzymes that are essential for spindle assembly, ciliogenesis, cell division, and cellular motility. We have recently shown that a related protein, Katanin-like 2 (KATNAL2), is similarly required for cytokinesis, cell cycle progression, and ciliogenesis in cultured mouse cells. However, its developmental expression pattern, localization, and in vivo role during organogenesis have yet to be characterized. Here, we used Xenopus embryos to reveal that Katnal2 (1) is expressed broadly in ciliated and neurogenic tissues throughout embryonic development; (2) is localized to basal bodies, ciliary axonemes, centrioles, and mitotic spindles; and (3) is required for ciliogenesis and brain development. Since human KATNAL2 is a risk gene for autism spectrum disorders, our functional data suggest that Xenopus may be a relevant system for understanding the relationship of mutations in this gene to autism and the underlying molecular mechanisms of pathogenesis.

Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

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.

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.

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.