Péter Lénárt

Oocyte meiosis is a specialized form of cell division adapted to produce a single haploid egg for fertilization. Diverse actin-driven mechanisms have essential roles in supporting these highly asymmetric divisions of the large oocyte. Whether these represent exotic adaptations in individual species or whether actin has broadly conserved functions in animal oocytes remains unclear. To address this, we established live-imaging assays, combined with targeted perturbations and biophysical modeling, in the non-bilaterian jellyfish Clytia hemisphaerica . We show that in Clytia, a nuclear F-actin network stabilizes the large oocyte nucleus. Upon meiotic entry, a transient F-actin shell forms to facilitate nuclear envelope rupture, followed by chromosome congression driven by the collapse of the nuclear F-actin network and capture by microtubules. Finally, the forming spindle is transported to the cell periphery by cytoplasmic flows produced by a wave of cortical contraction. Together, the presence of these actin-driven mechanisms in a basal metazoan evidence their ancient origin, establishing a metazoan complement of conserved molecular modules required for oocyte divisions.

Cell division involves the complete reorganization of the cellular architecture, requiring precise coordination of cellular processes in space and time, but how does this control system work at the atomic level? Three new studies offer a glimpse under the hood, revealing structural details of the intricate interactions between Aurora A and PLK1 kinases and the co-factor Bora, and how they act together to trigger timely entry to mitosis. Three new studies reveal structural details of the intricate interactions between Aurora A and PLK1 kinases and the co-factor Bora, and how they act together to trigger timely entry to mitosis.

The traditional microscopy workflow involves manual selection of objects one after the other. This is not only time consuming, but inevitably introduces human bias. Here, we describe a workflow, AutoSTED for identification of cell nuclei on wide-field fluorescence images and subsequent automated imaging of these nuclei at super-resolution by Stimulated Emission Depletion (STED) microscopy. We demonstrate that in an overnight run we are able to image hundreds of cells selected without bias. The resolution afforded by STED allowed straightforward identification and counting of individual nuclear pore complexes (NPCs), and enabled us to automatically extract parameters such as NPC density under different experimental conditions. Together, we illustrate how automation enables optimal use of instrument time to produce large unbiased datasets.

The Mos kinase activates the ERK/MAPK pathway during oocyte meiosis, controlling essential meiotic functions in species across metazoa. However, despite its significance, the molecular targets of Mos–MAPK remain largely unidentified. Here, we addressed this question using starfish oocytes ideally suited to combine cellular assays with phosphoproteomics. This revealed CPE-mediated mRNA polyadenylation as a prominent target of Mos–MAPK, and we show that translation is required to drive the second meiotic division. Secondly, we identify a well-defined subset of cytoskeletal regulators as targets of Mos–MAPK. We show that this regulation is critical to ensure the asymmetry of meiotic divisions, primarily by reducing the growth of astral microtubules. This allows positioning of the spindle directly beneath the cortex and prevents the separation of spindle poles in anaphase, thereby minimizing polar body size. Thus, by phosphoproteomics, we reveal molecular modules controlled by Mos–MAPK, explaining how this single, conserved kinase can act as a switch between the mitotic and meiotic division programs.

Polo-like kinase 1 (PLK1) is a conserved regulator of cell division. During mitotic prophase, PLK1 contributes to the activation of the cyclin-dependent kinase 1 (CDK1). However, the exact functions of PLK1 in prophase remain incompletely understood. Here, we show that PLK1 inhibition in synchronous G2 cell populations of multiple mammalian cell lines delays or prevents mitotic entry with high variability between individual cells. Using a mathematical model, we recapitulate this phenomenon and provide an explanation for the observed phenotypic variability. We show that PLK1-inhibited cells are delayed in a prophase-like state with low CDK1 activity that increases slowly and gradually over hours. These cells display progressively condensing chromosomes, increased microtubule dynamics, and reorganization of the actin cortex, while the nuclear envelope remains intact. We characterize this state further by phosphoproteomics, revealing phosphorylation of regulators of chromatin organization and the cytoskeleton consistent with the cellular phenotypes. Together, our results indicate that PLK1 inhibition stabilizes cells in a prophase-like state with low CDK1 activity displaying a specific set of early mitotic phosphorylation events.

The mechanical properties of the cytoplasm and nucleoplasm are crucial for the correct and robust functioning of a cell and play a key role in understanding how mechanical signals are transferred to the nucleus. Here, we demonstrate remarkable shape mimicry between the cellular and nuclear shape of oocytes, following the externally applied deformation without direct contact between the cell cortex and the nucleus. This effect arises from a surprisingly soft and fluid-like nucleoplasm that is barely resisting any external strain, while the viscoelastic cytoplasm drives shape transmission. Comparative studies in jellyfish, starfish, and mouse oocytes reveal that lower cytoplasmic elasticity in jellyfish leads to reduced nuclear shape mimicry, highlighting the role of cytoplasmic mechanics in nuclear deformation. Significance Statement: Mechanosensing of the nucleus is, in addition to chemical signalling, an important factor in gene expression. Although nuclei are often thought to be rigid inclusions in the cytoplasm of a cell, we show that in oocytes nuclei are much more deformable. Using a combination of intranuclear, intracellular and extracellular measurements, we attribute our findings to a fine balance between the soft nucleoplasm surrounded by an elastic shell and the viscoelastic properties of the cytoplasm.

Polo-like kinase 1 (plk1) is a conserved regulator of cell division. During prophase, plk1 phosphorylates direct substrates and is involved in activation of the cyclin-dependent kinase 1 (cdk1). However, the exact functions of plk1 in prophase remain incompletely understood. By testing several cell lines and small-molecule inhibitors, we confirm that plk1 inhibition causes a delay in mitotic entry. We show that cells are delayed in a prophase-like state displaying progressively condensing chromosomes, increased microtubule dynamics, reorganization of the actin cortex, while the nuclear envelope remains intact. We show that during this prolonged prophase cdk1 activity increases gradually over several hours with individual cells stochastically reaching the entry threshold, explaining the highly variable timing of mitotic entry. We then use phosphoproteomics to characterize this prolonged prophase state revealing for the first time phosphosites specific to prophase including several regulators of chromatin organization and the cytoskeleton. Together, we show that plk1 functions as a catalyst of prophase to prometaphase transition, and by using plk1 inhibition as a tool, we identify early changes in the phosphoproteome as the cell prepares for division.

The Mos kinase activates the ERK/MAPK pathway during oocyte meiosis, controlling essential meiotic functions in species across metazoa. However, despite its significance, the molecular targets of Mos-MAPK remain largely unidentified. Here, we addressed this question using starfish oocytes ideally suited to combine cellular assays with phosphoproteomics. This revealed CPE-mediated mRNA polyadenylation as a prominent target of Mos-MAPK, and we show that translation is required to drive the second meiotic division. Secondly, we identify a well-defined subset of cytoskeletal regulators as targets of Mos-MAPK. We show that this regulation is critical to ensure the asymmetry of meiotic divisions primarily by reducing the growth of astral microtubules. This allows positioning of the spindle directly beneath the cortex and prevents the separation of spindle poles in anaphase, thereby minimizing polar body size. Thus, by phosphoproteomics we reveal molecular modules controlled by Mos-MAPK explaining how this single, conserved kinase can act as a switch between the mitotic and meiotic division programs.

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin ‘shell’ in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells.