Electrochemical control of cell and tissue polarity.

Localized ion fluxes at the plasma membrane provide electrochemical gradients at the cell surface that contribute to cell polarization, migration, and division. Ion transporters, local pH gradients, membrane potential, and organization are emerging as important factors in cell polarization mechanisms. The power of electrochemical effects is illustrated by the ability of exogenous electric fields to redirect polarization in cells ranging from bacteria, fungi, and amoebas to keratocytes and neurons. Electric fields normally surround cells and tissues and thus have been proposed to guide cell polarity in development, cancer, and wound healing. Recent studies on electric field responses in model systems and development of new biosensors provide new avenues to dissect molecular mechanisms. Here, we review recent advances that bring molecular understanding of how electrochemistry contributes to cell polarity in various contexts.

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.

Influence of cell geometry on division-plane positioning.

The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces.

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.

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.

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.

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.

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.

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis.

The orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.

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.

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.

How cells sense their own shape - mechanisms to probe cell geometry and their implications in cellular organization and function.

Cells come in a variety of shapes that most often underlie their functions. Regulation of cell morphogenesis implies that there are mechanisms for shape sensing that still remain poorly appreciated. Global and local cell geometry features, such as aspect ratio, size or membrane curvature, may be probed by intracellular modules, such as the cytoskeleton, reaction-diffusion systems or molecular complexes. In multicellular tissues, cell shape emerges as an important means to transduce tissue-inherent chemical and mechanical cues into intracellular organization. One emergent paradigm is that cell-shape sensing is most often based upon mechanisms of self-organization, rather than determinism. Here, we review relevant work that has elucidated some of the core principles of how cellular geometry may be conveyed into spatial information to guide processes, such as polarity, signaling, morphogenesis and division-plane positioning.

Electrochemical regulation of budding yeast polarity.

Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization.

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.

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.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

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.