Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata.

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellate remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the two flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located: a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules: polar acetylated microtubules whose length is constant and central tyrosinated microtubules which elongate during chromosome segregation. During cell division a microtubule rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism independent of the actomyosin ring typical of animal and yeast cells.

Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos and Non-model Organisms.

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.

Acquisition of the spindle assembly checkpoint and its modulation by cell fate and cell size in a chordate embryo.

The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.

Daily variations of Ostreopsis cf. ovata abundances in NW Mediterranean Sea.

Ostreopsis cf. ovata is a benthic dinoflagellate very common in tropical and temperate coastal areas, particularly in the Mediterranean Sea. This species is also found in the plankton, i.e. swimming in the water column or in aggregates floating at the sea surface. The potential links between the planktonic and benthic populations influencing their relative distribution in the water column and attached to the benthic substrate are poorly understood. To shed light on this question, a high-frequency temporal monitoring was conducted in the Villefranche bay (France) to determine the abundance of (1) epibenthic cells attached to macroalgae, (2) planktonic cells in the water column and (3) cells in aggregates floating at the sea water surface (hereafter, referred to sea surface cells) . This monitoring was realized over 3 consecutive years (2018, 2019 and 2020) and at different phases of the bloom (exponential phase - 2020, peak - 2019 and decline phase - 2018). Strong variations in benthic and planktonic O. cf. ovata abundances were observed over the 24 h sampling cycles conducted in three consecutive years. The three populations, planktonic, benthic and sea surface cells, exhibited the highest numbers during the day (light) hours and lowest values at night in 2018 and 2019. In 2020, however, benthic abundances did not differ significantly between light and dark periods. Moreover, epibenthic cells abundances peaked in the morning, followed by the peak of the cells in the plankton and in the surface aggregates during the afternoon. Monitoring of O. cf. ovata is often based on a single sampling per day without precise indications of sampling time and shows great variability in O. cf. ovata abundances. Our observations of daily variations in cell abundances along the water column clearly indicate that time and water column depth of sampling constitute a great source of variability and have to be considered when designing new monitoring strategies to reduce variability and to harmonize data acquisition and international comparisons.

The Spindle Assembly Checkpoint Functions during Early Development in Non-Chordate Embryos.

In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of Xenopus and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.

Mechanical and molecular basis for the symmetrical division of the fission yeast nuclear envelope.

In fission yeast Schizosaccharomyces pombe, the nuclear envelope remains intact throughout mitosis and undergoes a series of symmetrical morphological changes when the spindle pole bodies (SPBs), embedded in the nuclear envelope, are pushed apart by elongating spindle microtubules. These symmetrical membrane shape transformations do not correspond to the shape behavior of an analogous system based on lipid vesicles. Here we report that the symmetry of the dividing fission yeast nucleus is ensured by SPB-chromosome attachments, as loss of kinetochore clustering in the vicinity of SPBs results in the formation of abnormal asymmetric shapes with long membrane tethers. We integrated these findings in a biophysical model, which explains the symmetry of the nuclear shapes on the basis of forces exerted by chromosomes clustered at SPBs on the extending nuclear envelope. Based on this analysis we conclude that the fission yeast nuclear envelope exhibits the same mechanical properties as simple lipid vesicles, but interactions with other cellular components, such as chromosomes, influence the nuclear shape during mitosis, allowing the formation of otherwise energetically unfavorable symmetrical dumbbell structures upon spindle elongation. The model allows us to explain the appearance of abnormal asymmetric shapes in fission yeast mutants with mis-segregated chromosomes as well as with altered nuclear membrane composition.

Growth phenotype screening of Schizosaccharomyces pombe using a Lensless microscope.

The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously. A simple algorithm to measure intensity changes in the Airy Disc First Fringe (ADFF) has been derived to follow the growth characteristics of the unicellular yeast Schizosaccharomyces pombe. The performance of the algorithm is calibrated using comparison between optical image and ADFF analysis of polystyrene microspheres with known dimensions and has an accuracy of 5% over all lengths above the diffraction-limited measurements. We have observed the growth characteristics of S. pombe for N=100 cells to determine the growth phenotype distributions of Length (L(t=0)) and width (W(t=0)) on arrival at the surface, lag phase adjustment to the new growth conditions (B), the length at birth, LB, and cell cycle length, tcell. The observed cell width distribution has a median width of 3.9 (±0.1) µm, as expected, but a non-normal distribution. Similarly, all growth parameters studied, L(t=0), LB and cell cycle time are phenotypes with non-normal distributions but with medians consistent with the literature values.

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier.

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.

Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

Microtubules offset growth site from the cell centre in fission yeast.

The design principles that underlie cellular morphogenetic mechanisms are central to understanding the generation of cell form. We have investigated the constraints governing the formation and positioning of new growth zones in the fission yeast cell and have shown that establishment of a new axis of polarity is independent of microtubules and that in the absence of microtubules a new growth zone is activated near the nucleus in the middle of the cell. Activation of a new growth zone can occur at any stage of the cell cycle as long as the nucleus is a sufficient distance away from previously growing ends. The positioning of growth zones is regulated by the polarity marker Tea1 delivered by microtubules; cells with short microtubules locate the growth zone near the region where the microtubules terminate. We propose a model for the activation of new growth zones comprising a long-range laterally inhibitory component and a self-activating positive local component that is delivered to cell ends by Tea1 and the microtubules. The principle of this symmetry-breaking design may also apply to the morphogenesis of other cells.

End4/Sla2 is involved in establishment of a new growth zone in Schizosaccharomyces pombe.

The rod-shaped Schizosaccharomyces pombe cell grows in a polarized fashion from opposing ends. Correct positioning of the growth zones is directed by the polarity marker Tea1 located at the cell ends where actin patches accumulate and cell growth takes place. We show that the S. pombe homologue of Saccharomyces cerevisiae SLA2, a protein involved in cortical actin organization and endocytosis, provides a link between the polarity marker and the growth machinery. In wild-type fission yeast cells, this homologue End4/Sla2 is enriched at cell ends during interphase and localizes to a medial ring at cell division, mirroring the actin localization pattern throughout the cell cycle. Proper localization relies on membrane trafficking and is independent of both the actin and microtubule cytoskeletons. End4/Sla2 is required for the establishment of new polarised growth zones, and deletion of its C-terminal talin-like domain prevents the establishment of a new growth zone after cell fission. We propose that End4/Sla2 acts downstream of the polarity marker Tea1 and is implicated in the recruitment of the actin cytoskeleton to bring about polarised cell growth.

Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte.

During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. We show that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome BRE-mediated repression. Moreover, we show that accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. As posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. We show that flies bearing the weak mutation orb(mel) localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. We conclude that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation.