Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much effort has been made to better understand the molecular mechanisms responsible for apical constriction, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging and tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance of the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

F-Actin Bending Facilitates Net Actomyosin Contraction By Inhibiting Expansion With Plus-End-Located Myosin Motors

We investigate whether a microscopic system of two semi-flexible actin filaments with an attached myosin motor can facilitate contraction. Based on energy minimisation, we derive and analyse a partial differential equation model for a two-filament-motor structure embedded within a dense, two-dimensional network. Our method enables calculation of the plane stress tensor, providing a measure for contractility. After deriving the model, we use a combination of asymptotic analysis and numerical solutions to show how F-actin bending facilitates net contraction as a myosin motor traverses two symmetric filaments. Myosin motors close to the minus-ends facilitate contraction, whereas motors close to the plus-ends facilitate expansion. The leading-order solution for rigid filaments exhibits polarity-reversal symmetry, such that the contractile and expansive components balance to zero. Surprisingly, after introducing bending the first-order correction to stress indicates expansion. However, numerical solutions show that filament bending induces a geometric asymmetry that brings the filaments closer to parallel as a myosin motor approaches their plus-ends. This decreases the effective spring force opposing motion of the motor, enabling it to move faster close to filament plus-ends. This reduces the contribution of expansive stress, giving rise to net contraction. Further numerical solutions confirm that this applies beyond the small bending regime considered in the asymptotic analysis. Our findings confirm that filament bending gives rise to microscopic-scale actomyosin contraction, and provides a possible explanation for network-scale contraction.

LFA-1 and Kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB), and SKAP-55 (SKAP1) are recruited into microclusters and activate integrins via the effectors Talin-1 and Kindlin-3. We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity, and dynein motor function. Although immobilized VLA-4 (a4b1) and LFA-1 (aLb2) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin b2, Kindlin-3, and Zyxin are required for complete centripetal transport, while integrin b1 and Talin-1 are not. CD69 upregulation is similarly dependent on integrin b2, Kindlin-3, and Zyxin, but not Talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and Kindlin-3.

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2–5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II–mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.

Role of the polycystins as mechanosensors of extracellular stiffness

Polycystin-1 has recently emerged as a possible receptor able to sense extracellular stiffness and to negatively control the cellular actomyosin contraction machinery. Here, we revisit a large body of literature on autosomal dominant polycystic kidney disease providing a new possible mechanistic view on the topic.

Calcium waves facilitate and coordinate the contraction of endfeet actin stress fibers in Drosophila interommatidial cells

Actomyosin contraction shapes the Drosophila eye’s panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. Here, we discover a novel phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC Ca2+ waves are independent of phototransduction, but require inositol 1,4,5-triphosphate receptor (IP3R), suggesting these waves are mediated by Ca2+ releases from ER stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitating stress fiber contraction and floor morphogenesis. Further, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating ECM and its interaction with stress fibers in eye morphogenesis. We propose that coordinated Ca2+ spikes in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor.Summary StatementCa2+ waves have an important role in generating tensile forces to shape the Drosophila eye’s convex curvature. Coordinated Ca2+ spikes facilitate actomyosin contractions at the basal endfeet of interommatidial cells.

Collagen I triggers directional migration, invasion and matrix remodeling of stroma cells in a 3D spheroid model of endometriosis

Endometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.

Nitric oxide controls excitatory/inhibitory balance in the hypoglossal nucleus during early postnatal development

Synaptic remodeling during early postnatal development lies behind neuronal networks refinement and nervous system maturation. In particular, the respiratory system is immature at birth and is subjected to significant postnatal development. In this context, the excitatory/inhibitory balance dramatically changes in the respiratory-related hypoglossal nucleus (HN) during the 3 perinatal weeks. Since, development abnormalities of hypoglossal motor neurons (HMNs) are associated with sudden infant death syndrome and obstructive sleep apnea, deciphering molecular partners behind synaptic remodeling in the HN is of basic and clinical relevance. Interestingly, a transient expression of the neuronal isoform of nitric oxide (NO) synthase (NOS) occurs in HMNs at neonatal stage that disappears before postnatal day 21 (P21). NO, in turn, is a determining factor for synaptic refinement in several physiopathological conditions. Here, intracerebroventricular chronic administration (P7–P21) of the broad spectrum NOS inhibitor l-NAME (N(ω)-nitro-l-arginine methyl ester) differentially affected excitatory and inhibitory rearrangement during this neonatal interval in the rat. Whilst l-NAME led to a reduction in the number of excitatory structures, inhibitory synaptic puncta were increased at P21 in comparison to administration of the inactive stereoisomer d-NAME. Finally, l-NAME decreased levels of the phosphorylated form of myosin light chain in the nucleus, which is known to regulate the actomyosin contraction apparatus. These outcomes indicate that physiologically synthesized NO modulates excitatory/inhibitory balance during early postnatal development by acting as an anti-synaptotrophic and/or synaptotoxic factor for inhibitory synapses, and as a synaptotrophin for excitatory ones. The mechanism of action could rely on the modulation of the actomyosin contraction apparatus.