Mechanobiology: Shaping the future of cellular form and function.

Mechanobiology-the field studying how cells produce, sense, and respond to mechanical forces-is pivotal in the analysis of how cells and tissues take shape in development and disease. As we venture into the future of this field, pioneers share their insights, shaping the trajectory of future research and applications.

Robust axis elongation by Nodal-dependent restriction of BMP signaling.

Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.

Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization.

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole-a protuberance of the zygote's vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.

Adhesion-induced cortical flows pattern E-cadherin-mediated cell contacts.

Metazoan development relies on the formation and remodeling of cell-cell contacts. Dynamic reorganization of adhesion receptors and the actomyosin cell cortex in space and time plays a central role in cell-cell contact formation and maturation. Nevertheless, how this process is mechanistically achieved when new contacts are formed remains unclear. Here, by building a biomimetic assay composed of progenitor cells adhering to supported lipid bilayers functionalized with E-cadherin ectodomains, we show that cortical F-actin flows, driven by the depletion of myosin-2 at the cell contact center, mediate the dynamic reorganization of adhesion receptors and cell cortex at the contact. E-cadherin-dependent downregulation of the small GTPase RhoA at the forming contact leads to both a depletion of myosin-2 and a decrease of F-actin at the contact center. At the contact rim, in contrast, myosin-2 becomes enriched by the retraction of bleb-like protrusions, resulting in a cortical tension gradient from the contact rim to its center. This tension gradient, in turn, triggers centrifugal F-actin flows, leading to further accumulation of F-actin at the contact rim and the progressive redistribution of E-cadherin from the contact center to the rim. Eventually, this combination of actomyosin downregulation and flows at the contact determines the characteristic molecular organization, with E-cadherin and F-actin accumulating at the contact rim, where they are needed to mechanically link the contractile cortices of the adhering cells.

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

ZnUMBA - a live imaging method to detect local barrier breaches.

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance, which measures ion flux across a monolayer, or by adding traceable macromolecules and monitoring their passage across the monolayer. Although these methods measure changes in global barrier function, they lack the sensitivity needed to detect local or transient barrier breaches, and they do not reveal the location of barrier leaks. Therefore, we previously developed a method that we named the zinc-based ultrasensitive microscopic barrier assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction leaks with high spatiotemporal resolution. Here, we present expanded applications for ZnUMBA. ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin accumulation following laser injury. ZnUMBA can also be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby canine kidney (MDCK) II epithelial cells. ZnUMBA is a powerful and flexible method that, with minimal optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.

A hydraulic feedback loop between mesendoderm cell migration and interstitial fluid relocalization promotes embryonic axis formation in zebrafish.

Interstitial fluid (IF) accumulation between embryonic cells is thought to be important for embryo patterning and morphogenesis. Here, we identify a positive mechanical feedback loop between cell migration and IF relocalization and find that it promotes embryonic axis formation during zebrafish gastrulation. We show that anterior axial mesendoderm (prechordal plate [ppl]) cells, moving in between the yolk cell and deep cell tissue to extend the embryonic axis, compress the overlying deep cell layer, thereby causing IF to flow from the deep cell layer to the boundary between the yolk cell and the deep cell layer, directly ahead of the advancing ppl. This IF relocalization, in turn, facilitates ppl cell protrusion formation and migration by opening up the space into which the ppl moves and, thereby, the ability of the ppl to trigger IF relocalization by pushing against the overlying deep cell layer. Thus, embryonic axis formation relies on a hydraulic feedback loop between cell migration and IF relocalization.

Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin localization in Ciona.

Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.

Multitier mechanics control stromal adaptations in the swelling lymph node.

Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion.

Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells.

Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow [J.-L. Maître et al., Science 338, 253-256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension-stabilizing E-cadherin-actin complexes at the contact.

Rigidity transitions in development and disease.

Although rigidity and jamming transitions have been widely studied in physics and material science, their importance in a number of biological processes, including embryo development, tissue homeostasis, wound healing, and disease progression, has only begun to be recognized in the past few years. The hypothesis that biological systems can undergo rigidity/jamming transitions is attractive, as it would allow these systems to change their material properties rapidly and strongly. However, whether such transitions indeed occur in biological systems, how they are being regulated, and what their physiological relevance might be, is still being debated. Here, we review theoretical and experimental advances from the past few years, focussing on the regulation and role of potential tissue rigidity transitions in different biological processes.

Combined effect of cell geometry and polarity domains determines the orientation of unequal division.

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).

Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis.

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

Dissecting Organismal Morphogenesis by Bridging Genetics and Biophysics.

Multicellular organisms develop complex shapes from much simpler, single-celled zygotes through a process commonly called morphogenesis. Morphogenesis involves an interplay between several factors, ranging from the gene regulatory networks determining cell fate and differentiation to the mechanical processes underlying cell and tissue shape changes. Thus, the study of morphogenesis has historically been based on multidisciplinary approaches at the interface of biology with physics and mathematics. Recent technological advances have further improved our ability to study morphogenesis by bridging the gap between the genetic and biophysical factors through the development of new tools for visualizing, analyzing, and perturbing these factors and their biochemical intermediaries. Here, we review how a combination of genetic, microscopic, biophysical, and biochemical approaches has aided our attempts to understand morphogenesis and discuss potential approaches that may be beneficial to such an inquiry in the future.

Apical contacts stemming from incomplete delamination guide progenitor cell allocation through a dragging mechanism.

The developmental strategies used by progenitor cells to allow a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here, we uncovered a mechanism of progenitor cell allocation that stems from an incomplete process of epithelial delamination that allows progenitors to coordinate their movement with adjacent extra-embryonic tissues. Progenitors of the zebrafish laterality organ originate from the superficial epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-lasting apical contacts that enable the epithelial layer to pull a subset of progenitors on their way to the vegetal pole. The remaining delaminated cells follow the movement of apically attached progenitors by a protrusion-dependent cell-cell contact mechanism, avoiding sequestration by the adjacent endoderm, ensuring their collective fate and allocation at the site of differentiation. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.

Holding it together: when cadherin meets cadherin.

Intercellular adhesion is the key to multicellularity, and its malfunction plays an important role in various developmental and disease-related processes. Although it has been intensively studied by both biologists and physicists, a commonly accepted definition of cell-cell adhesion is still being debated. Cell-cell adhesion has been described at the molecular scale as a function of adhesion receptors controlling binding affinity, at the cellular scale as resistance to detachment forces or modulation of surface tension, and at the tissue scale as a regulator of cellular rearrangements and morphogenesis. In this review, we aim to summarize and discuss recent advances in the molecular, cellular, and theoretical description of cell-cell adhesion, ranging from biomimetic models to the complexity of cells and tissues in an organismal context. In particular, we will focus on cadherin-mediated cell-cell adhesion and the role of adhesion signaling and mechanosensation therein, two processes central for understanding the biological and physical basis of cell-cell adhesion.

Rigidity percolation uncovers a structural basis for embryonic tissue phase transitions.

Embryo morphogenesis is impacted by dynamic changes in tissue material properties, which have been proposed to occur via processes akin to phase transitions (PTs). Here, we show that rigidity percolation provides a simple and robust theoretical framework to predict material/structural PTs of embryonic tissues from local cell connectivity. By using percolation theory, combined with directly monitoring dynamic changes in tissue rheology and cell contact mechanics, we demonstrate that the zebrafish blastoderm undergoes a genuine rigidity PT, brought about by a small reduction in adhesion-dependent cell connectivity below a critical value. We quantitatively predict and experimentally verify hallmarks of PTs, including power-law exponents and associated discontinuities of macroscopic observables. Finally, we show that this uniform PT depends on blastoderm cells undergoing meta-synchronous divisions causing random and, consequently, uniform changes in cell connectivity. Collectively, our theoretical and experimental findings reveal the structural basis of material PTs in an organismal context.

Quantifying Tissue Tension in the Granulosa Layer After Laser Surgery.

Tissue morphogenesis is driven by mechanical forces triggering cell movements and shape changes. Quantitatively measuring tension within tissues is of great importance for understanding the role of mechanical signals acting on the cell and tissue level during morphogenesis. Here we introduce laser ablation as a useful tool to probe tissue tension within the granulosa layer, an epithelial monolayer of somatic cells that surround the zebrafish female gamete during folliculogenesis. We describe in detail how to isolate follicles, mount samples, perform laser surgery, and analyze the data.