Biogenesis and function of ESCRT-dependent extracellular vesicles.

From bacteria to humans, cells secrete a large variety of membrane-bound extracellular vesicles. Only relatively recently has it however started to become clear that the exovesicular transport of proteins and RNAs is important for normal physiology and numerous pathological conditions. Extracellular vesicles can be formed through the release of the intralumenal vesicles of multivesicular endosomes as so-called exosomes, or through direct, ectosomal, budding from the cell surface. Through their ability to promote the bending of membranes away from the cytoplasm, the components of the Endosomal Sorting Complex Required for Transport (ESCRT) have been implicated in both exo- and ectosomal biogenesis. Studies of the ESCRT machinery may therefore provide important insights into the formation and function of extracellular vesicles. In the present review, we first describe the cell biological mechanisms through which ESCRT components contribute to the biogenesis of different types of extracellular vesicles. We then discuss how recent functional studies have started to uncover important roles of ESCRT-dependent extracellular vesicles in a wide variety of processes, including the transport of developmental signaling molecules and embryonic morphogenesis, the regulation of social behavior and host-pathogen interactions, as well as the etiology and progression of neurodegenerative pathologies and cancer.

The ESCRT machinery regulates the secretion and long-range activity of Hedgehog.

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

Systems dynamics in endocytosis.

The endocytic system acts at the crossroads of different cellular activities to play a central role in the regulation of cell signaling and membrane dynamics. An European Molecular Biology Organization (EMBO) conference held in October 2013 in Villars-sur-Ollon gathered researchers from all over the world to present their latest findings on the endolysosomal system and identify major challenges for the future. The conference covered the entire spectrum of research in this rapidly evolving field ranging from the cellular mechanics of endocytosis to the role of proteins and lipids in the biogenesis and function of endolysosomal organelles and the analysis of higher order system properties in multicellular contexts. In particular, the meeting highlighted current efforts to complement the insights that can be gained by biochemical and cell biological approaches with the use of quantitative biophysics, systems biology and animal model systems to achieve an integrated view of the properties of the endomembrane system and its role in cellular information processing.

Myosin1G promotes Nodal signaling to control zebrafish left-right asymmetry.

Myosin1D (Myo1D) has recently emerged as a conserved regulator of animal Left-Right (LR) asymmetry that governs the morphogenesis of the vertebrate central LR Organizer (LRO). In addition to Myo1D, the zebrafish genome encodes the closely related Myo1G. Here we show that while Myo1G also controls LR asymmetry, it does so through an entirely different mechanism. Myo1G promotes the Nodal-mediated transfer of laterality information from the LRO to target tissues. At the cellular level, Myo1G is associated with endosomes positive for the TGFß signaling adapter SARA. myo1g mutants have fewer SARA-positive Activin receptor endosomes and a reduced responsiveness to Nodal ligands that results in a delay of left-sided Nodal propagation and tissue-specific laterality defects in organs that are most distant from the LRO. Additionally, Myo1G promotes signaling by different Nodal ligands in specific biological contexts. Our findings therefore identify Myo1G as a context-dependent regulator of the Nodal signaling pathway.

The evolutionarily conserved choroid plexus contributes to the homeostasis of brain ventricles in zebrafish.

The choroid plexus (ChP) produces cerebrospinal fluid (CSF). It also contributes to brain development and serves as the CSF-blood barrier. Prior studies have identified transporters on the epithelial cells that transport water and ions from the blood vasculature to the ventricles and tight junctions involved in the CSF-blood barrier. Yet, how the ChP epithelial cells control brain physiology remains unresolved. We use zebrafish to provide insights into the physiological roles of the ChP. Upon histological and transcriptomic analyses, we identify that the zebrafish ChP is conserved with mammals and expresses transporters involved in CSF secretion. Next, we show that the ChP epithelial cells secrete proteins into CSF. By ablating the ChP epithelial cells, we identify a reduction of the ventricular sizes without alterations of the CSF-blood barrier. Altogether, our findings reveal that the zebrafish ChP is conserved and contributes to the size and homeostasis of the brain ventricles.

The E3 ubiquitin ligase mindbomb1 controls planar cell polarity-dependent convergent extension movements during zebrafish gastrulation.

Vertebrate Delta/Notch signaling involves multiple ligands, receptors and transcription factors. Delta endocytosis - a critical event for Notch activation - is however essentially controlled by the E3 Ubiquitin ligase Mindbomb1 (Mib1). Mib1 inactivation is therefore often used to inhibit Notch signaling. However, recent findings indicate that Mib1 function extends beyond the Notch pathway. We report a novel Notch-independent role of Mib1 in zebrafish gastrulation. mib1 null mutants and morphants display impaired Convergence Extension (CE) movements. Comparison of different mib1 mutants and functional rescue experiments indicate that Mib1 controls CE independently of Notch. Mib1-dependent CE defects can be rescued using the Planar Cell Polarity (PCP) downstream mediator RhoA, or enhanced through knock-down of the PCP ligand Wnt5b. Mib1 regulates CE through its RING Finger domains that have been implicated in substrate ubiquitination, suggesting that Mib1 may control PCP protein trafficking. Accordingly, we show that Mib1 controls the endocytosis of the PCP component Ryk and that Ryk internalization is required for CE. Numerous morphogenetic processes involve both Notch and PCP signaling. Our observation that during zebrafish gastrulation Mib1 exerts a Notch-independent control of PCP-dependent CE movements suggest that Mib1 loss-of-function phenotypes should be cautiously interpreted depending on the biological context.

Animal embryonic development involves producing an entire animal from a single starting cell, the zygote. To do this, the zygote must divide to make new cells, and these cells have to arrange themselves into the correct body shape. This requires a lot of cells to move in a coordinated fashion. One of these movements is called 'convergent extension', in which a typically round group of cells rearranges into a long, thin shape, for example, to increase the distance between the head and the tail of the animal. In order to coordinate this movement, cells need to communicate with each other. One of the signaling pathways cells use to guide them to the right positions is the planar cell polarity (PCP) pathway. Zebrafish are used to study PCP in convergent extension because they are transparent, making it easy to track their cell movements under the microscope. Interestingly, when a protein called Mindbomb1 (Mib1) is inactivated in zebrafish embryos, convergent extension is reduced. Mib1 helps control the activity of other proteins by attaching a chemical marker called ubiquitin to them, which tags these proteins to be relocated from the cell surface to small vesicles within the cell. The protein is known to be involved in the formation of neurons ­ the cells that make up the brain and nerves ­ but its links to cell movement and the PCP pathway had not been explored. Saraswathy et al. used a technique called Crispr/Cas9 mutagenesis to genetically modify zebrafish and then used observations under the microscope to determine the role of Mib1 in PCP and convergent extension. Their experiments show that Mib1 helps internalize a protein called Ryk from the cell surface into the cell. This internalization of Ryk is required to relay signals through the PCP pathway. When Mib1 is missing, Ryk stays on the surface of the cell, instead of moving to the inside, blocking PCP signaling between cells and therefore blocking convergent extension. Understanding the role of Mib1 in PCP signaling sheds light on how cell movements are coordinated during the embryonic development of zebrafish. Future research will involve determining whether Mib1 plays the same role in other animals, offering further insights into embryonic development. Additionally, PCP is known to have a role in disease, including the spread of cancer. It will be important to determine whether Mib1 is involved in this process as well.

Endocytic regulation of notch signalling during development.

Delta/Notch signalling is of major importance for embryonic development and adult life. While endocytosis is often viewed as a way to down-regulate biological signals, ligand and receptor internalization are essential for Notch activation. The development of Drosophila mecanosensory bristles is a powerful model to study Delta/Notch signalling. Following the asymmetric division of bristle precursor cells, Delta ligands and Notch receptors traffic differently in the two daughter cells, leading to directional signal activation. Recent evidence suggests that in addition to differential ligand endocytosis after division, a subpopulation of multivesicular endosomes ensures the directional transport of Delta/Notch already during asymmetric cell division. Biochemical analysis suggests that different phases of endocytic Delta trafficking exert complementary but distinct actions required for ligand recycling, ligand/receptor interaction and ligand-mediated receptor activation, respectively. Finally, novel data suggest that different endosomal compartments may act as Delta/Notch signalling platforms. In this review, we discuss the implications of these novel findings for our cell biological understanding of Delta/Notch signalling.

Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling.

Fibroblast growth factors (FGFs) are pleiotrophic growth factors that control cell proliferation, migration, differentiation and embryonic patterning. During early zebrafish embryonic development, FGFs regulate dorsoventral patterning by controlling ventral bone morphogenetic protein (BMP) expression. FGFs function by binding and activating high-affinity tyrosine kinase receptors. FGF activity is negatively regulated by members of the Sprouty family, which antagonize Ras signalling induced by receptor tyrosine kinases. On the basis of similarities in their expression patterns during embryonic development, we have identified five genes that define a synexpression group -- fgf8, fgf3, sprouty2, sprouty4, as well as a novel gene, sef (similar expression to fgf genes). Sef encodes a conserved putative transmembrane protein that shares sequence similarities with the intracellular domain of the interleukin 17 receptor. Here we show that in zebrafish, Sef functions as a feedback-induced antagonist of Ras/Raf/MEK/MAPK-mediated FGF signalling.

Functional Analysis of ESCRT-Positive Extracellular Vesicles in the Drosophila Wing Imaginal Disc.

A large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. In this chapter, we describe a series of experimental approaches that we established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh).Through the combined use of genetic, cell biological, and imaging approaches, we investigate four important aspects of exovesicle biology: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands.

Notch-mediated inhibition of neurogenesis is required for zebrafish spinal cord morphogenesis.

The morphogenesis of the nervous system requires coordinating the specification and differentiation of neural precursor cells, the establishment of neuroepithelial tissue architecture and the execution of specific cellular movements. How these aspects of neural development are linked is incompletely understood. Here we inactivate a major regulator of embryonic neurogenesis - the Delta/Notch pathway - and analyze the effect on zebrafish central nervous system morphogenesis. While some parts of the nervous system can establish neuroepithelial tissue architecture independently of Notch, Notch signaling is essential for spinal cord morphogenesis. In this tissue, Notch signaling is required to repress neuronal differentiation and allow thereby the emergence of neuroepithelial apico-basal polarity. Notch-mediated suppression of neurogenesis is also essential for the execution of specific morphogenetic movements of zebrafish spinal cord precursor cells. In the wild-type neural tube, cells divide at the organ midline to contribute one daughter cell to each organ half. Notch signaling deficient animals fail to display this behavior and therefore form a misproportioned spinal cord. Taken together, our findings show that Notch-mediated suppression of neurogenesis is required to allow the execution of morphogenetic programs that shape the zebrafish spinal cord.

Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry.

The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. Here, we show that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2), to shape a productive LRO flow. Our findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

[The ESCRT complex: from endosomal transport to the development of multicellular organisms]. / Le complexe ESCRT : du transport endosomal au développement d'organismes multicellulaires.

Since its discovery more than 50 years ago, the endo-lysosomal system has emerged as a central integrator of different cellular activities. This vesicular trafficking apparatus governs processes as diverse as the transduction of stimuli by growth factor receptors, the recycling and secretion of signaling molecules and the regulation of cellular homeostasis through autophagy. Accordingly, dysfunctions of the vesicular transport machinery have been linked to a growing number of pathologies. In this review we take the "Endosomal Sorting Complex Required for Transport" (ESCRT) as an example to illustrate the multiple functions of an evolutionarily conserved endosomal transport machinery. We describe the major concepts that have emerged from the study of this machinery at the level of the development and the physiology of multi-cellular organisms. In particular, we highlight the essential contributions of ESCRT proteins on the regulation of three biological processes: the endocytic regulation of cell signaling, autophagy and its role in neuronal morphogenesis and finally the biogenesis and function of extracellular vesicles.

Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.

Asymmetric division of neural precursor cells contributes to the generation of a variety of neuronal types. Asymmetric division is mediated by the asymmetric inheritance of fate determinants by the two daughter cells. In vertebrates, asymmetric fate determinants, such as Par3 and Mib, are only now starting to be identified. Here we show that, during mitosis of neural precursors in zebrafish, directional trafficking of Sara endosomes to one of the daughters can function as such a determinant. In asymmetric lineages, where one daughter cell becomes a neuron (n cell) whereas the other divides again to give rise to two neurons (p cell), we found that the daughter that inherits most of the Sara endosomes acquires the p fate. Sara endosomes carry an endocytosed pool of the Notch ligand DeltaD, which is thereby itself distributed asymmetrically. Sara and Notch are both essential for cell fate assignation within asymmetric lineages. Therefore, the Sara endosome system determines the fate decision between neuronal differentiation and mitosis in asymmetric lineages and thereby contributes to controlling the number of neural precursors and differentiated neurons during neurogenesis in a vertebrate.

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division.

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis [1-6]. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate [1-6]. We have previously shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter [6]. Here we show that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. Our data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis.

Report of the Second European Zebrafish Principal Investigator Meeting in Karlsruhe, Germany, March 21-24, 2012.

The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.

Endocytosis and mitosis: a two-way relationship.

Cell division involves a vast remodelling of cellular membranes. This is most apparent for the cell surface, but it is also true for internal vesicular organelles such as the Golgi apparatus. while the contribution of endocytosis to membrane trafficking and signal processing in interphase cells is well established, the role of the endocytic system in cell division has long been neglected. A number of recent studies have however shed novel light on this issue. Here, we review findings supporting the existence of two important links between endocytosis and mitosis: First, endocytic trafficking is essential to reshape the plasma membrane during cell division. Second, cell division affects the partitioning, the trafficking and hence the activity of the signalling molecules that are contained within endocytic compartments.