Cell-intrinsic and -extrinsic roles of the ESCRT-III subunit Shrub in abscission of Drosophila sensory organ precursors.

Although the molecular mechanisms governing abscission of isolated cells have largely been elucidated, those underlying the abscission of epithelial progenitors surrounded by epidermal cells (ECs), connected via cellular junctions, remain largely unexplored. Here, we investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursors (SOPs). We found that SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJ assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to quicker disentanglement of neighboring cell membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for the transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions and is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. Our study uncovers cell-intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission.

The Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila sensory organ precursors cells.

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics.

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of γ-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.

Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo.

In Drosophila, the sensory organ precursor (SOP or pI cell) divides asymmetrically to give birth to daughter cells, the fates of which are governed by the differential activation of the Notch pathway. Proteolytic activation of Notch induced by ligand is based on the correct polarized sorting and localization of the Notch ligand Delta, the Notch receptor and its trafficking partner Sanpodo (Spdo). Here, we have identified Stratum (Strat), a presumptive guanine nucleotide exchange factor for Rab GTPases, as a regulator of Notch activation. Loss of Strat causes cell fate transformations associated with an accumulation of Notch, Delta and Spdo in the trans-Golgi network (TGN), and an apical accumulation of Spdo. The strat mutant phenotype is rescued by the catalytically active as well as the wild-type form of Rab8, suggesting a chaperone function for Strat rather than that of exchange factor. Strat is required to localize Rab8 at the TGN, and rab8 phenocopies strat We propose that Strat and Rab8 act at the exit of the Golgi apparatus to regulate the sorting and the polarized distribution of Notch, Delta and Spdo.

Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium.

Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion.

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth.

Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs' anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.

Epithelial cell division - multiplying without losing touch.

Epithelia are compact tissues comprising juxtaposed cells that function as mechanical and chemical barriers between the body and the environment. This barrier relies, in part, on adhesive contacts within adherens junctions, which are formed and stabilized by E-cadherin and catenin proteins linked to the actomyosin cytoskeleton. During development and throughout adult life, epithelia are continuously growing or regenerating, largely as a result of cell division. Although persistence of adherens junctions is needed for epithelial integrity, these junctions are continually remodelled during cell division. In this Commentary, we will focus on cytokinesis, the final step of mitosis, a multiparty phenomenon in which the adherens junction belt plays an essential role and during which a new cell-cell interface is generated between daughter cells. This new interface is the site of intense remodelling, where new adhesive contacts are assembled and cell polarity is transmitted from mother to daughter cells, ultimately becoming the site of cell signalling.

Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes.

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells.

Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary cell fate acquisition following asymmetric cell division.

Notch signalling is involved in numerous cellular processes during development and throughout adult life. Although ligands and receptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissues and is accurately regulated in time and space. Previous studies have demonstrated that endocytosis and recycling of both ligands and/or receptors are essential for this regulation. However, the precise endocytic routes, compartments and regulators involved in the spatiotemporal regulation are largely unknown. In order to identify intracellular trafficking regulators of Notch signalling, we have undertaken a tissue-specific dsRNA genetic screen of candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in the Drosophila melanogaster sensory organ lineage in which Notch signalling regulates binary cell fate acquisition. Gain or loss of Notch signalling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes were found to regulate the steady state localisation of Notch, Sanpodo, a Notch co-factor, and/or Delta in the pupal lineage. In particular, we identified 20 genes with previously unknown function in D. melanogaster intracellular trafficking. Among them, we identified CG2747 and we show that it regulates the localisation of clathrin adaptor AP-1 complex, a negative regulator of Notch signalling. Together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch-signalling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands during signalling.

ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of septate junction integrity in Drosophila epithelial cells.

Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum, alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, ß-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector ß-Heavy Spectrin Karst, and ß-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.

The multiple facets of ubiquitination in the regulation of notch signaling pathway.

The Notch signaling pathway regulates numerous aspects of metazoan development and tissue renewal. Deregulation or loss of Notch signaling is associated with a wide range of human disorders from developmental syndromes to cancer. Notch receptors and their ligands are widely expressed throughout development, yet Notch activation is robustly controlled in a spatio-temporal manner. Within the past decades, genetic screens and biochemical approaches led to the identification of more than 10 E3 ubiquitin ligases and deubiquitinating enzymes implicated in the regulation of the Notch pathway. In this review, we highlight the recent studies in Notch signaling that reveal how ubiquitination of components of the Notch pathway, ranging from degradation to regulation of membrane trafficking, impacts on the developmental control of the signaling activities of both Notch receptors and their ligands.

Regulation of Notch signalling by endocytosis and endosomal sorting.

Cell-cell signalling is an essential process in the formation of multicellular organisms. Notch is the receptor of an evolutionarily conserved signalling pathway regulating numerous developmental decisions. Indeed, its misregulation is linked to multiple developmental and physiological disorders. Notch and its ligands are distributed widely throughout development, yet Notch activity is highly controlled and restricted in time and space. Recent advances have highlighted that endocytosis followed by endosomal sorting of both the Notch receptor and its ligands is an essential mechanism by which Notch-mediated signalling is developmentally controlled.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells.

In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. Here, we report that the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. We propose that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling.

Mitochondrial Aurora kinase A induces mitophagy by interacting with MAP1LC3 and Prohibitin 2.

Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex and restores normal ATP production levels. Altogether, these data provide evidence for a role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.

Super-resolved live-cell imaging using random illumination microscopy.

Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Interplay between Anakonda, Gliotactin, and M6 for Tricellular Junction Assembly and Anchoring of Septate Junctions in Drosophila Epithelium.

In epithelia, tricellular junctions (TCJs) serve as pivotal sites for barrier function and integration of both biochemical and mechanical signals [1-3]. In Drosophila, TCJs are composed of the transmembrane protein Sidekick at the adherens junction (AJ) level, which plays a role in cell-cell contact rearrangement [4-6]. At the septate junction (SJ) level, TCJs are formed by Gliotactin (Gli) [7], Anakonda (Aka) [8, 9], and the Myelin proteolipid protein (PLP) M6 [10, 11]. Despite previous data on TCJ organization [12-14], TCJ assembly, composition, and links to adjacent bicellular junctions (BCJs) remain poorly understood. Here, we have characterized the making of TCJs within the plane of adherens junctions (tricellular adherens junction [tAJ]) and the plane of septate junctions (tricellular septate junction [tSJ]) and report that their assembly is independent of each other. Aka and M6, whose localizations are interdependent, act upstream to localize Gli. In turn, Gli stabilizes Aka at tSJ. Moreover, tSJ components are not only essential at vertex, as we found that loss of tSJ integrity induces micron-length bicellular SJ (bSJ) deformations. This phenotype is associated with the disappearance of SJ components at tricellular contacts, indicating that bSJs are no longer connected to tSJs. Reciprocally, SJ components are required to restrict the localization of Aka and Gli at vertex. We propose that tSJs function as pillars to anchor bSJs to ensure the maintenance of tissue integrity in Drosophila proliferative epithelia.

Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.

The use of the semiconductor quantum dots (QD) as biolabels for both ensemble and single-molecule tracking requires the development of simple and versatile methods to target individual proteins in a controlled manner, ideally in living cells. To address this challenge, we have prepared small and stable QDs (QD-ND) using a surface coating based on a peptide sequence containing a tricysteine, poly(ethylene glycol) (PEG), and an aspartic acid ligand. These QDs, with a hydrodynamic diameter of 9 +/- 1.5 nm, can selectively bind to polyhistidine-tagged (histag) proteins in vitro or in living cells. We show that the small and monodisperse size of QD-ND allows for the formation of QD-ND/histag protein complexes of well-defined stoichiometry and that the 1:1 QD/protein complex can be isolated and purified by gel electrophoresis without any destabilization in the nanomolar concentration range. We also demonstrate that QD-ND can be used to specifically label a membrane receptor with an extracellular histag expressed in living HeLa cells. Here, cytotoxicity tests reveal that cell viability remains high under the conditions required for cellular labeling with QD-ND. Finally, we apply QD-ND complexed with histag end binding protein-1 (EB1), a microtubule associated protein, to single-molecule tracking in Xenopus extracts. Specific colocalization of QD-ND/EB1 with microtubules during the mitotic spindle formation demonstrates that QD-ND and our labeling strategy provide an efficient approach to monitor the dynamic behavior of proteins involved in complex biological functions.

Unequal segregation of Neuralized biases Notch activation during asymmetric cell division.

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). We report that the E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. We propose that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling.

[Sorting, recycling and WNT signaling: Wntless and retromer functions]. / Tri sélectif et recyclage: Wntless et le retromer, deux acteurs clés de la signalisation WNT.

Cell-cell signaling is essential for the development of multi-cellular organisms. Indeed, membrane traffic is required for the correct sorting and function of receptors and ligands. In the past decades, many genetic screens performed in C. elegans and Drosophila have been crucial to identify the role of intracellular traffic in signaling. In this review, we discuss recent work that led to the identification of Wntless, a sorting receptor for WNT, and of the retromer, a protein complex required for the recycling of Wntless from endosomes to the trans-Golgi network.