FREEDA: An automated computational pipeline guides experimental testing of protein innovation.

Cell biologists typically focus on conserved regions of a protein, overlooking innovations that can shape its function over evolutionary time. Computational analyses can reveal potential innovations by detecting statistical signatures of positive selection that lead to rapid accumulation of beneficial mutations. However, these approaches are not easily accessible to non-specialists, limiting their use in cell biology. Here, we present an automated computational pipeline FREEDA that provides a simple graphical user interface requiring only a gene name; integrates widely used molecular evolution tools to detect positive selection in rodents, primates, carnivores, birds, and flies; and maps results onto protein structures predicted by AlphaFold. Applying FREEDA to >100 centromere proteins, we find statistical evidence of positive selection within loops and turns of ancient domains, suggesting innovation of essential functions. As a proof-of-principle experiment, we show innovation in centromere binding of mouse CENP-O. Overall, we provide an accessible computational tool to guide cell biology research and apply it to experimentally demonstrate functional innovation.

CDC6 as a Key Inhibitory Regulator of CDK1 Activation Dynamics and the Timing of Mitotic Entry and Progression.

Timely mitosis is critically important for early embryo development. It is regulated by the activity of the conserved protein kinase CDK1. The dynamics of CDK1 activation must be precisely controlled to assure physiologic and timely entry into mitosis. Recently, a known S-phase regulator CDC6 emerged as a key player in mitotic CDK1 activation cascade in early embryonic divisions, operating together with Xic1 as a CDK1 inhibitor upstream of the Aurora A and PLK1, both CDK1 activators. Herein, we review the molecular mechanisms that underlie the control of mitotic timing, with special emphasis on how CDC6/Xic1 function impacts CDK1 regulatory network in the Xenopus system. We focus on the presence of two independent mechanisms inhibiting the dynamics of CDK1 activation, namely Wee1/Myt1- and CDC6/Xic1-dependent, and how they cooperate with CDK1-activating mechanisms. As a result, we propose a comprehensive model integrating CDC6/Xic1-dependent inhibition into the CDK1-activation cascade. The physiological dynamics of CDK1 activation appear to be controlled by the system of multiple inhibitors and activators, and their integrated modulation ensures concomitantly both the robustness and certain flexibility of the control of this process. Identification of multiple activators and inhibitors of CDK1 upon M-phase entry allows for a better understanding of why cells divide at a specific time and how the pathways involved in the timely regulation of cell division are all integrated to precisely tune the control of mitotic events.

FREEDA: an automated computational pipeline guides experimental testing of protein innovation by detecting positive selection.

Cell biologists typically focus on conserved regions of a protein, overlooking innovations that can shape its function over evolutionary time. Computational analyses can reveal potential innovations by detecting statistical signatures of positive selection that leads to rapid accumulation of beneficial mutations. However, these approaches are not easily accessible to non-specialists, limiting their use in cell biology. Here, we present an automated computational pipeline FREEDA (Finder of Rapidly Evolving Exons in De novo Assemblies) that provides a simple graphical user interface requiring only a gene name, integrates widely used molecular evolution tools to detect positive selection, and maps results onto protein structures predicted by AlphaFold. Applying FREEDA to >100 mouse centromere proteins, we find evidence of positive selection in intrinsically disordered regions of ancient domains, suggesting innovation of essential functions. As a proof-of-principle experiment, we show innovation in centromere binding of CENP-O. Overall, we provide an accessible computational tool to guide cell biology research and apply it to experimentally demonstrate functional innovation.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing "HURP-gap". The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.

Centromere drive: model systems and experimental progress.

Centromeres connect chromosomes and spindle microtubules to ensure faithful chromosome segregation. Paradoxically, despite this conserved function, centromeric DNA evolves rapidly and centromeric proteins show signatures of positive selection. The centromere drive hypothesis proposes that centromeric DNA can act like a selfish genetic element and drive non-Mendelian segregation during asymmetric female meiosis. Resulting fitness costs lead to genetic conflict with the rest of the genome and impose a selective pressure for centromeric proteins to adapt by suppressing the costs. Here, we describe experimental model systems for centromere drive in yellow monkeyflowers and mice, summarize key findings demonstrating centromere drive, and explain molecular mechanisms. We further discuss efforts to test if centromeric proteins are involved in suppressing drive-associated fitness costs, highlight a model for centromere drive and suppression in mice, and put forth outstanding questions for future research.

Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions.

Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.

Spindle-Length-Dependent HURP Localization Allows Centrosomes to Control Kinetochore-Fiber Plus-End Dynamics.

During mitosis, centrosomes affect the length of kinetochore fibers (k-fibers) and the stability of kinetochore-microtubule attachments, implying that they regulate k-fiber dynamics. However, the exact cellular and molecular mechanisms of this regulation remain unknown. Here, we created human cells with only one centrosome to investigate these mechanisms. Such cells formed asymmetric bipolar spindles that resulted in asymmetric cell divisions. K-fibers in the acentrosomal half-spindles were shorter, more stable, and had a reduced poleward microtubule flux at minus ends and more frequent pausing events at their plus ends. This indicates that centrosomes regulate k-fiber dynamics both locally at minus ends and far away at plus ends. At the molecular level, we find that the microtubule-stabilizing protein HURP is enriched on the k-fiber plus ends in the acentrosomal half-spindles of cells with only one centrosome. HURP depletion rebalances k-fiber stability and plus-end dynamics in such cells and improves spindle and cell division symmetry. Our data from 3 different cell lines indicate that HURP accumulates on k-fibers inversely proportionally to half-spindle length. We therefore propose that centrosomes regulate k-fiber plus ends indirectly via length-dependent accumulation of HURP.

Complete microtubule-kinetochore occupancy favours the segregation of merotelic attachments.

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.

Symmetry Does not Come for Free: Cellular Mechanisms to Achieve a Symmetric Cell Division.

During mitosis cells can divide symmetrically to proliferate or asymmetrically to generate tissue diversity. While the mechanisms that ensure asymmetric cell division have been extensively studied, it is often assumed that a symmetric cell division is the default outcome of mitosis. Recent studies, however, imply that the symmetric nature of cell division is actively controlled, as they reveal numerous mechanisms that ensure the formation of equal-sized daughter cells as cells progress through cell division. Here we review our current knowledge of these mechanisms and highlight possible key questions in the field.

The equatorial position of the metaphase plate ensures symmetric cell divisions.

Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore-microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

Control of timing of embryonic M-phase entry and exit is differentially sensitive to CDK1 and PP2A balance.

Harmonious embryo development requires precise coordination between the timing of the cell cycle and the developmental program. Cyclin accumulation determines the timing of the cell cycle M-phase entry and its degradation determines the timing of the M-phase exit. It is well known that CDK1 and PP2A also govern M-phase entry. However, it is unknown how this kinase and phosphatase regulate the precise timing of events at the beginning of the M-phase and how they cooperate with cyclin metabolism. Here we use Xenopus laevis one-cell embryo cell-free extract experiments to answer this question critical for understanding the regulation of embryo development. Using, separately, low concentrations of the chemical inhibitor of CDK1, RO3306 (RO), or the inhibitor of phosphatases, okadaic acid (OA), we show that moderately diminished CDK1 or PP2A activities results in a delay and an acceleration respectively, of M-phase entry. Simultaneous diminution of CDK1 and PP2A activities results in an intermediate timing of M-phase entry, prolongs the duration of M-phase and diminishes the dynamics of cyclin B2 degradation. We thus show, for the first time, that equilibrium between CDK1 and PP2A specifies the timing of M-phase entry and exit and regulates the dynamics of cyclin B degradation upon M-phase exit in Xenopus laevis first embryonic mitosis.