Delayed abscission in animal cells - from development to defects.

Cell division involves separating the genetic material and cytoplasm of a mother cell into two daughter cells. The last step of cell division, abscission, consists of cutting the cytoplasmic bridge, a microtubule-rich membranous tube connecting the two cells, which contains the midbody, a dense proteinaceous structure. Canonically, abscission occurs 1-3 h after anaphase. However, in certain cases, abscission can be severely delayed or incomplete. Abscission delays can be caused by mitotic defects that activate the abscission 'NoCut' checkpoint in tumor cells, as well as when cells exert abnormally strong pulling forces on the bridge. Delayed abscission can also occur during normal organism development. Here, we compare the mechanisms triggering delayed and incomplete abscission in healthy and disease scenarios. We propose that NoCut is not a bona fide cell cycle checkpoint, but a general mechanism that can control the dynamics of abscission in multiple contexts.

Active mesh and neural network pipeline for cell aggregate segmentation.

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.

A quick, cheap, and reliable protocol for immunofluorescence of pluripotent and differentiating mouse embryonic stem cells in 2D and 3D colonies.

Immunofluorescent labeling is a widely used method to visualize endogenous proteins. It can be expensive and difficult to stain mouse embryonic stem cells (mESCs) because they require expensive growth media, prefer specific substrates, grow in 3D, and have loose cell-substrate adhesion. Here we propose a half-a-day, cheap, easy-to-follow, and reproducible protocol for immunofluorescence of mESCs. This protocol has been streamlined to allow a fast visualization of the investigated proteins, and we provide tips specific to stem cell culture. For complete details on the use and execution of this protocol, please refer to Chaigne et al. (2021).1.

Incomplete abscission and cytoplasmic bridges in the evolution of eukaryotic multicellularity.

The textbook view of cell division terminates with the final separation of the two daughter cells in the process called abscission. However, in contrast to this classical view, a variety of cell types in multicellular organisms are connected through cytoplasmic bridges, which most often form by incomplete abscission or - more rarely - by local fusion of plasma membranes. In this review, we survey the distribution, function, and formation of cytoplasmic bridges across the eukaryotic tree of life. We find that cytoplasmic bridges are widespread, and were likely ancestrally present, in almost all lineages of eukaryotes with clonal multicellularity - including the five 'complex multicellular' lineages: animals, fungi, land plants, red algae, and brown algae. In animals, cytoplasmic bridges resulting from incomplete abscission are ubiquitous in the germline and common in pluripotent cell types. Although cytoplasmic bridges have been less studied than other structural mediators of multicellularity (such as adhesion proteins and extracellular matrix), we propose that they have played a pivotal role in the repeated evolution of eukaryotic clonal multicellularity - possibly by first performing a structural role and later by allowing exchange of nutrients and/or intercellular communication, which notably buffered cell-cell competition by averaging gene expression. Bridges were eventually lost from many animal tissues in concert with the evolution of spatial cell differentiation, cell motility within the organism, and other mechanisms for intercellular distribution of signals and metabolites. Finally, we discuss the molecular basis for the evolution of incomplete abscission and examine the alternative hypotheses of single or multiple origins.

Cell state transitions: definitions and challenges.

A fundamental challenge when studying biological systems is the description of cell state dynamics. During transitions between cell states, a multitude of parameters may change - from the promoters that are active, to the RNAs and proteins that are expressed and modified. Cells can also adopt different shapes, alter their motility and change their reliance on cell-cell junctions or adhesion. These parameters are integral to how a cell behaves and collectively define the state a cell is in. Yet, technical challenges prevent us from measuring all of these parameters simultaneously and dynamically. How, then, can we comprehend cell state transitions using finite descriptions? The recent virtual workshop organised by The Company of Biologists entitled 'Cell State Transitions: Approaches, Experimental Systems and Models' attempted to address this question. Here, we summarise some of the main points that emerged during the workshop's themed discussions. We also present examples of cell state transitions and describe models and systems that are pushing forward our understanding of how cells rewire their state.

Three-dimensional geometry controls division symmetry in stem cell colonies.

Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we use mouse embryonic stem cells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell-cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry. This article has an associated First Person interview with the first author of the paper.

Abscission Couples Cell Division to Embryonic Stem Cell Fate.

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cell types, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here, we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs naive pluripotency exit, and artificially inducing abscission accelerates it. Altogether, our data indicate that a switch in the division machinery leading to faster abscission regulates pluripotency exit. Our study identifies abscission as a key cellular process coupling cell division to fate transitions.

Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.

Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.

Artificially decreasing cortical tension generates aneuploidy in mouse oocytes.

Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.

In Vitro Fertilization of Mouse Oocyte.

The early development of embryos is a widely studied process. Fertilization is the consequence of the fusion of two haploid gametes, the oocyte and the sperm. In mammals, such as human and mouse, this occurs in the female genital tracks. However, imaging in utero is still very limited, which prompts the use of extra utero techniques. In particular, in vitro fertilization provides a quick and simple way to study the very early steps of mouse embryo development. Here, I describe a simple and hands-on protocol to perform in vitro fertilization using the mouse as a model system.

Asymmetries and Symmetries in the Mouse Oocyte and Zygote.

Mammalian oocytes grow periodically after puberty thanks to the dialogue with their niche in the follicle. This communication between somatic and germ cells promotes the accumulation, inside the oocyte, of maternal RNAs, proteins and other molecules that will sustain the two gamete divisions and early embryo development up to its implantation. In order to preserve their stock of maternal products, oocytes from all species divide twice minimizing the volume of their daughter cells to their own benefit. For this, they undergo asymmetric divisions in size where one main objective is to locate the division spindle with its chromosomes off-centred. In this chapter, we will review how this main objective is reached with an emphasis on the role of actin microfilaments in this process in mouse oocytes, the most studied example in mammals. This chapter is subdivided into three parts: I-General features of asymmetric divisions in mouse oocytes, II-Mechanism of chromosome positioning by actin in mouse oocytes and III-Switch from asymmetric to symmetric division at the oocyte-to-embryo transition.

F-actin mechanics control spindle centring in the mouse zygote.

Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

A soft cortex is essential for asymmetric spindle positioning in mouse oocytes.

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

Spindle positioning in mammalian oocytes.

To preserve the maternal stores accumulated during oogenesis for further embryo development, oocytes divide asymmetrically which minimizes the volume of cytoplasm lost with each set of haploid genome. To ensure asymmetric division to occur, oocytes have to position their division spindle asymmetrically as well as tailor the size of daughter cells to the chromatin mass. In this review, we will discuss the recent advances in the field, with emphasis on the control mechanisms involved in meiotic spindle positioning in mammalian oocytes.