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.

Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers.

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.

Cytoplasm mechanics and cellular organization.

As cells organize spatially or divide, they translocate many micron-scale organelles in their cytoplasm. These include endomembrane vesicles, nuclei, microtubule asters, mitotic spindles, or chromosomes. Organelle motion is powered by cytoskeleton forces but is opposed by viscoelastic forces imparted by the surrounding crowded cytoplasm medium. These resistive forces associated to cytoplasm physcial properties remain generally underappreciated, yet reach significant values to slow down organelle motion or even limit their displacement by springing them back towards their original position. The cytoplasm may also be itself organized in time and space, being for example stiffer or more fluid at certain locations or during particular cell cycle phases. Thus, cytoplasm mechanics may be viewed as a labile module that contributes to organize cells. We here review emerging methods, mechanisms, and concepts to study cytoplasm mechanical properties and their function in organelle positioning, cellular organization and division.

Length limitation of astral microtubules orients cell divisions in murine intestinal crypts.

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

Size- and position-dependent cytoplasm viscoelasticity through hydrodynamic interactions with the cell surface.

Many studies of cytoplasm rheology have focused on small components in the submicrometer scale. However, the cytoplasm also baths large organelles like nuclei, microtubule asters, or spindles that often take significant portions of cells and move across the cytoplasm to regulate cell division or polarization. Here, we translated passive components of sizes ranging from few up to ~50 percents of the cell diameter, through the vast cytoplasm of live sea urchin eggs, with calibrated magnetic forces. Creep and relaxation responses indicate that for objects larger than the micron size, the cytoplasm behaves as a Jeffreys material, viscoelastic at short timescales, and fluidizing at longer times. However, as component size approached that of cells, cytoplasm viscoelastic resistance increased in a nonmonotonic manner. Flow analysis and simulations suggest that this size-dependent viscoelasticity emerges from hydrodynamic interactions between the moving object and the static cell surface. This effect also yields to position-dependent viscoelasticity with objects initially closer to the cell surface being harder to displace. These findings suggest that the cytoplasm hydrodynamically couples large organelles to the cell surface to restrain their motion, with important implications for cell shape sensing and cellular organization.

Regulation and functions of cell division in the intestinal tissue.

In multicellular organisms, epithelial cells are key elements of tissue organization. In developing epithelial tissues, cellular proliferation and differentiation are under the tight regulation of morphogenetic programs to ensure correct organ formation and functioning. In these processes, proliferation rates and division orientation regulate the speed, timing and direction of tissue expansion but also its proper patterning. Moreover, tissue homeostasis relies on spatio-temporal modulations of daughter cell behavior and arrangement. These aspects are particularly crucial in the intestine, which is one of the most proliferative tissues in adults, making it a very attractive adult organ system to study the role of cell division on epithelial morphogenesis and organ function. Although epithelial cell division has been the subject of intense research for many years in multiple models, it still remains in its infancy in the context of the intestinal tissue. In this review, we focus on the current knowledge on cell division and regulatory mechanisms at play in the intestinal epithelial tissue, as well as their importance in developmental biology and physiopathology.

Cell wall dynamics stabilize tip growth in a filamentous fungus.

Hyphal tip growth allows filamentous fungi to colonize space, reproduce, or infect. It features remarkable morphogenetic plasticity including unusually fast elongation rates, tip turning, branching, or bulging. These shape changes are all driven from the expansion of a protective cell wall (CW) secreted from apical pools of exocytic vesicles. How CW secretion, remodeling, and deformation are modulated in concert to support rapid tip growth and morphogenesis while ensuring surface integrity remains poorly understood. We implemented subresolution imaging to map the dynamics of CW thickness and secretory vesicles in Aspergillus nidulans. We found that tip growth is associated with balanced rates of CW secretion and expansion, which limit temporal fluctuations in CW thickness, elongation speed, and vesicle amount, to less than 10% to 20%. Affecting this balance through modulations of growth or trafficking yield to near-immediate changes in CW thickness, mechanics, and shape. We developed a model with mechanical feedback that accounts for steady states of hyphal growth as well as rapid adaptation of CW mechanics and vesicle recruitment to different perturbations. These data provide unprecedented details on how CW dynamics emerges from material secretion and expansion, to stabilize fungal tip growth as well as promote its morphogenetic plasticity.

Mechanobiology of the cell wall - insights from tip-growing plant and fungal cells.

The cell wall (CW) is a thin and rigid layer encasing the membrane of all plant and fungal cells. It ensures mechanical integrity by bearing mechanical stresses derived from large cytoplasmic turgor pressure, contacts with growing neighbors or growth within restricted spaces. The CW is made of polysaccharides and proteins, but is dynamic in nature, changing composition and geometry during growth, reproduction or infection. Such continuous and often rapid remodeling entails risks of enhanced stress and consequent damages or fractures, raising the question of how the CW detects and measures surface mechanical stress and how it strengthens to ensure surface integrity? Although early studies in model fungal and plant cells have identified homeostatic pathways required for CW integrity, recent methodologies are now allowing the measurement of pressure and local mechanical properties of CWs in live cells, as well as addressing how forces and stresses can be detected at the CW surface, fostering the emergence of the field of CW mechanobiology. Here, using tip-growing cells of plants and fungi as case study models, we review recent progress on CW mechanosensation and mechanical regulation, and their implications for the control of cell growth, morphogenesis and survival.

An interpretable and versatile machine learning approach for oocyte phenotyping.

Meiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.

Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning.

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics, and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division, remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original positions. These forces are independent of cytoskeletal force generators yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Cells under pressure: how yeast cells respond to mechanical forces.

In their natural habitats, unicellular fungal microbes are exposed to a myriad of mechanical cues such as shear forces from fluid flow, osmotic changes, and contact forces arising from microbial expansion in confined niches. While the rigidity of the cell wall is critical to withstand such external forces and balance high internal turgor pressure, it poses mechanical challenges during physiological processes such as cell growth, division, and mating that require cell wall remodeling. Thus, even organisms as simple as yeast have evolved complex signaling networks to sense and respond to intrinsic and extrinsic mechanical forces. In this review, we summarize the type and origin of mechanical forces experienced by unicellular yeast and discuss how these forces reorganize cell polarity and how pathogenic fungi exploit polarized assemblies to track weak spots in host tissues for successful penetration. We then describe mechanisms of force-sensing by conserved sets of mechanosensors. Finally, we elaborate downstream mechanotransduction mechanisms that orchestrate appropriate cellular responses, leading to improved mechanical fitness.

Cell division geometries as central organizers of early embryo development.

Early cellular patterning is a critical step of embryonic development that determines the proper progression of morphogenesis in all metazoans. It relies on a series of rapid reductive divisions occurring simultaneously with the specification of the fate of different subsets of cells. Multiple species developmental strategies emerged in the form of a unique cleavage pattern with stereotyped division geometries. Cleavage geometries have long been associated to the emergence of canonical developmental features such as cell cycle asynchrony, zygotic genome activation and fate specification. Yet, the direct causal role of division positioning on blastomere cell behavior remain partially understood. Oriented and/or asymmetric divisions define blastomere cell sizes, contacts and positions, with potential immediate impact on cellular decisions, lineage specification and morphogenesis. Division positions also instruct daughter cells polarity, mechanics and geometries, thereby influencing subsequent division events, in an emergent interplay that may pattern early embryos independently of firm deterministic genetic programs. We here review the recent literature which helped to delineate mechanisms and functions of division positioning in early embryos.

Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.

Developing cardiovascular systems use mechanical forces to take shape, but how ubiquitous blood flow forces instruct local cardiac cell identity is still unclear. By manipulating mechanical forces in vivo, we show here that shear stress is necessary and sufficient to promote valvulogenesis. We found that valve formation is associated with the activation of an extracellular adenosine triphosphate (ATP)­dependent purinergic receptor pathway, specifically triggering calcium ion (Ca2+) pulses and nuclear factor of activated T cells 1 (Nfatc1) activation. Thus, mechanical forces are converted into discrete bioelectric signals by an ATP-Ca2+-Nfatc1­mechanosensitive pathway to generate positional information and control valve formation.

Detection of surface forces by the cell-wall mechanosensor Wsc1 in yeast.

Surface receptors of animal cells, such as integrins, promote mechanosensation by forming clusters as signaling hubs that transduce tensile forces. Walled cells of plants and fungi also feature surface sensors, with long extracellular domains that are embedded in their cell walls (CWs) and are thought to detect injuries and promote repair. How these sensors probe surface forces remains unknown. By studying the conserved CW sensor Wsc1 in fission yeast, we uncovered the formation of micrometer-sized clusters at sites of force application onto the CW. Clusters assembled within minutes of CW compression, in dose dependence with mechanical stress and disassembled upon relaxation. Our data support that Wsc1 accumulates to sites of enhanced mechanical stress through reduced lateral diffusivity, mediated by the binding of its extracellular WSC domain to CW polysaccharides, independent of canonical polarity, trafficking, and downstream CW regulatory pathways. Wsc1 may represent an autonomous module to detect and transduce local surface forces onto the CW.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

An image analysis method to survey the dynamics of polar protein abundance in the regulation of tip growth.

Tip growth is critical for the lifestyle of many walled cells. In yeast and fungi, this process is typically associated with the polarized deposition of conserved tip factors, including landmarks, Rho GTPases, cytoskeleton regulators, and membrane and cell wall remodelers. Because tip growth speeds may vary extensively between life cycles or species, we asked whether the local amount of specific polar elements could determine or limit tip growth speeds. Using the model fission yeast, we developed a quantitative image analysis pipeline to dynamically correlate single tip elongation speeds and polar protein abundance in large data sets. We found that polarity landmarks are typically diluted by growth. In contrast, tip growth speed is positively correlated with the local amount of factors related to actin, secretion or cell wall remodeling, but, surprisingly, exhibits long saturation plateaus above certain concentrations of those factors. Similar saturation observed for Spitzenkörper components in much faster growing fungal hyphae suggests that elements independent of canonical surface remodelers may limit single tip growth. This work provides standardized methods and resources to decipher the complex mechanisms that control cell growth.This article has an associated First Person interview with Sarah Taheraly, joint first author of the paper.

In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium.

The forces generated by microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on MT force exertion by motors anchored to a static surface, such as the cell cortex in vivo or glass surfaces in vitro [2-4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization, and orientation [5-14] but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo sizes and medium viscosities shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study reinforces the notion that endomembrane transport can exert significant forces on MTs.

The Perinuclear ER Scales Nuclear Size Independently of Cell Size in Early Embryos.

Nuclear size plays pivotal roles in gene expression, embryo development, and disease. A central hypothesis in organisms ranging from yeast to vertebrates is that nuclear size scales to cell size. This implies that nuclei may reach steady-state sizes set by limiting cytoplasmic pools of size-regulating components. By monitoring nuclear dynamics in early sea urchin embryos, we found that nuclei undergo substantial growth in each interphase, reaching a maximal size prior to mitosis that declined steadily over the course of development. Manipulations of cytoplasmic volume through multiple chemical and physical means ruled out cell size as a major determinant of nuclear size and growth. Rather, our data suggest that the perinuclear endoplasmic reticulum, accumulated through dynein activity, serves as a limiting membrane pool that sets nuclear surface growth rate. Partitioning of this local pool at each cell division modulates nuclear growth kinetics and dictates size scaling throughout early development.

Cytoskeleton Force Exertion in Bulk Cytoplasm.

The microtubule and actin cytoskeletons generate forces essential to position centrosomes, nuclei, and spindles for division plane specification. While the largest body of work has documented force exertion at, or close to the cell surface, mounting evidence suggests that cytoskeletal polymers can also produce significant forces directly from within the cytoplasm. Molecular motors such as kinesin or dynein may for instance displace cargos and endomembranes in the viscous cytoplasm yielding friction forces that pull or push microtubules. Similarly, the dynamics of bulk actin assembly/disassembly or myosin-dependent contractions produce cytoplasmic forces which influence the spatial organization of cells in a variety of processes. We here review the molecular and physical mechanisms supporting bulk cytoplasmic force generation by the cytoskeleton, their limits and relevance to organelle positioning, with a particular focus on cell division.

Systematic mapping of cell wall mechanics in the regulation of cell morphogenesis.

Walled cells of plants, fungi, and bacteria come with a large range of shapes and sizes, which are ultimately dictated by the mechanics of their cell wall. This stiff and thin polymeric layer encases the plasma membrane and protects the cells mechanically by opposing large turgor pressure derived mechanical stresses. To date, however, we still lack a quantitative understanding for how local and/or global mechanical properties of the wall support cell morphogenesis. Here, we combine subresolution imaging and laser-mediated wall relaxation to quantitate subcellular values of wall thickness (h) and bulk elastic moduli (Y) in large populations of live mutant cells and in conditions affecting cell diameter in the rod-shaped model fission yeast. We find that lateral wall stiffness, defined by the surface modulus, σ = hY, robustly scales with cell diameter. This scaling is valid across tens of mutants spanning various functions-within the population of individual isogenic strains, along single misshaped cells, and even across the fission yeasts clade. Dynamic modulations of cell diameter by chemical and/or mechanical means suggest that the cell wall can rapidly adapt its surface mechanics, rendering stretched wall portions stiffer than unstretched ones. Size-dependent wall stiffening constrains diameter definition and limits size variations; it may also provide an efficient means to keep elastic strains in the wall below failure strains, potentially promoting cell survival. This quantitative set of data impacts our current understanding of the mechanics of cell walls and its contribution to morphogenesis.