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In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis., During development, oriented cell division is important to proper body axis extension. Here, the authors show that sphingolipids are required to direct spindle rotation and oriented mitosis via Anthrax receptor palmitoylation in zebrafish gastrulation.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule gamma-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the down-stream mediators of Notch signaling, the Notch transcription acti-vation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB -103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lym-phoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrange-ments in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that in-terferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch -driven cancers.

Cells and organelles are delimited by lipid bilayers in which high deformability is essential to many cell processes, including motility, endocytosis and cell division. Membrane tension is therefore a major regulator of the cell processes that remodel membranes, albeit one that is very hard to measure in vivo. Here we show that a planarizable push-pull fluorescent probe called FliptR (fluorescent lipid tension reporter) can monitor changes in membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. The fluorescence lifetime depends linearly on membrane tension within cells, enabling an easy quantification of membrane tension by fluorescence lifetime imaging microscopy. We further show, using model membranes, that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension-dependent lipid phase separation. We also provide calibration curves that enable accurate measurement of membrane tension using fluorescence lifetime imaging microscopy.

Fig. 4 and the legend for Fig. 5 have been updated.

Cyclic oligochalcogenides are emerging as powerful tools to penetrate cells. With disulfide ring tension maximized, selenium chemistry had to be explored next to enhance speed and selectivity of dynamic covalent exchange on the way into the cytosol. We show that diseleno lipoic acid (DiSeL) delivers a variety of relevant substrates. DiSeL-driven uptake of artificial metalloenzymes enables bioorthogonal fluorophore uncaging within cells. Binding of a bicyclic peptide, phalloidin, to actin fibers evinces targeted delivery to the cytosol. Automated tracking of diffusive compared to directed motility and immobility localizes 79 % of protein-coated quantum dots (QDs) in the cytosol, with little endosomal capture (0.06 %). These results suggest that diselenolanes might act as molecular walkers along disulfide tracks in locally denatured membrane proteins, surrounded by adaptive micellar membrane defects. Miniscule and versatile, DiSeL tags are also readily available, stable, soluble, and non-toxic.

Nucleic acid templated reactions are enabled by the hybridization of probe-reagent conjugates resulting in high effective reagent concentration and fast chemical transformation. We have developed a reaction that harnesses cellular microRNA (miRNA) to yield the cleavage of a linker releasing fluorogenic rhodamine in a live vertebrate. The reaction is based on the catalytic photoreduction of an azide by a ruthenium complex. We showed that this system reports specific expression of miRNA in living tissues of a vertebrate., We have developed a reaction that harnesses cellular microRNA (miRNA) to yield the cleavage of a linker releasing fluorogenic rhodamine in a live vertebrate. The reaction is based on the catalytic photoreduction of an azide by a ruthenium complex.

Summary Secreted growth factors can act as morphogens that form spatial concentration gradients in developing organs, thereby controlling growth and patterning. For some morphogens, adaptation of the gradients to tissue size allows morphological patterns to remain proportioned as the organs grow. In the zebrafish pectoral fin, we found that BMP signaling forms a two-dimensional gradient. The length of the gradient scales with tissue length and its amplitude increases with fin size according to a power-law. Gradient scaling and amplitude power-laws are signatures of growth control by time derivatives of morphogenetic signaling: cell division correlates with the fold change over time of the cellular signaling levels. We show that Smoc1 regulates BMP gradient scaling and growth in the fin. Smoc1 scales the gradient by means of a feedback loop: Smoc1 is a BMP agonist and BMP signaling represses Smoc1 expression. Our work uncovers a layer of morphogen regulation during vertebrate appendage development., Graphical Abstract, Highlights • Zebrafish pectoral fins form two BMP signaling gradients that scale with fin size • The gradients show signatures of growth control by time derivatives of signaling • Smoc1 mutants fail to scale the BMP signaling gradient and have fin growth defects • A regulatory feedback loop Smoc1-BMP mediates BMP gradient scaling, The control of organ growth is essential for embryonic development. Mateus et al. show that during zebrafish pectoral fin development, two BMP morphogen signaling gradients form and expand proportionally to fin size, regulating growth. A feedback loop between Smoc1 (a conserved secreted factor) and BMP signaling mediates BMP gradient scaling.

We present a theory of pattern formation in growing domains inspired by biological examples of tissue development. Gradients of signaling molecules regulate growth, while growth changes these graded chemical patterns by dilution and advection. We identify a critical point of this feedback dynamics, which is characterized by spatially homogeneous growth and proportional scaling of patterns with tissue length. We apply this theory to the biological model system of the developing wing of the fruit fly \textit{Drosophila melanogaster} and quantitatively identify signatures of the critical point., 5 pages, 3 figures

Endocytosis is key in a number of cell events. In particular, its role during cell division has been a challenging question: while early studies examined whether endocytosis occurs during cell division, recent works show that, during division, cells do perform endocytosis actively. More importantly, during asymmetric cell division, endocytic pathways also control Notch signaling: endocytic vesicles regulate the presence, at the plasma membrane, of receptors and ligands at different levels between the two-daughter cells. Both early and late endocytic compartments have been shown to exert key regulatory controls by up-regulating or down-regulating Notch signaling in those cells. This biased Notch signaling enable finally cell fate assignation and specification which play a central role in development and physiology. In this chapter, we cover a number of significant works on endosomal trafficking evincing the importance of endocytosis in Notch-mediated cell fate specification during development.

How a developing organ grows and patterns to its final shape is an important question in developmental biology. Studies of growth and patterning in the Drosophila wing imaginal disc have identified a key player, the morphogen Decapentaplegic (Dpp). These studies provided insights into our understanding of growth control and scaling: expansion of the Dpp gradient correlated with the growth of the tissue. A recent report on growth of a Drosophila organ other than the wing, the eye imaginal disc, prompts a reconsideration of our models of growth control. Despite striking differences between the two, the Dpp gradient scales with the target tissues of both organs and the growth of both the wing and the eye is controlled by Dpp. The goal of this review is to discuss whether a parsimonious model of scaling and growth control can explain the relationship between the Dpp gradient and growth in these two different developmental systems.

Cell biologists now have tools and knowledge to generate useful quantitative data. But how can we make sense of these data, and are we measuring the correct parameters? Moreover, how can we test hypotheses quantitatively? To answer these questions, the theory of physics is required and is essential to the future of quantitative cell biology.

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. We show that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting., Asymmetric segregation of cell fate determinants during cell division governs daughter cell fate. Here the authors show that Sara endosomes, known to regulate Notch signalling, are targeted to the mitotic spindle and once phosphorylated are asymmetrically dispatched into a daughter cell to determine cell fate.

The Biochemistry Department at the University of Geneva currently has four full professors, a professor emeritus, one assistant professor, two MER (Maître d'enseignement et de Recherche) and a permanent scientific collaborator. The research interests of the members of the Biochemistry Department are described.

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol. Furthermore, these QDs can be functionalized, carrying GFP or anti-GFP nanobodies for instance. Our technology thus paves the way toward single molecule imaging in cells and living animals, allowing to probe biophysical properties of the cytosol.

During morphogenesis, organs grow to stereotyped sizes, but growth control mechanisms are poorly understood. Here, we measured the signaling dynamics of the morphogen Dpp, one of several Drosophila factors controlling morphogenetic growth, in the developing eye. In this tissue, the Dpp expression domain advances from the posterior to the anterior tissue edge. In front of this moving morphogen source, signaling inputs including Dpp activate the target gene hairy in a gradient that scales with tissue size. Proliferation, in turn, occurs in a mitotic wave in front of the source, whereas behind it, cells arrest and differentiate. We found that cells divide when their signaling levels have increased by around 60%. This simple mechanism quantitatively explains the proliferation and differentiation waves in wild type and mutants. Furthermore, this mechanism may be a common feature of different growth factors, because a Dpp-independent growth input also follows this growth rule.

The Notch signaling pathway plays important roles in many organisms and developmental contexts. The activities of the Notch receptor and of its ligand Delta are known to be regulated at several steps along the endocytic pathway. However, the precise molecular mechanism of Notch activation and the role played by endosomal sorting and trafficking remain elusive. We developed an antibody uptake assay to enable live imaging of endogenous internalized Notch and Delta in Drosophila tissues. In this chapter, we describe how to perform live antibody uptake assays in the Drosophila notum. In this tissue, Notch signaling plays a crucial role in the regulation of cell fate decisions in the lineage of sensory organ precursor (SOP) cells. We describe here how to do a correlative analysis of Notch/Delta live imaging in dividing SOPs and of the lineage of these particular SOPs. Combined with the wide range of genetic and chemical tools available in Drosophila research, these two methods will provide a better understanding of the role played by endocytic proteins and endosomal trafficking in Notch regulation, in terms of botch Notch trafficking and Notch signaling output.

Cells at different positions in a developing tissue receive different concentrations of signaling molecules, called morphogens, and this influences their cell fate. Morphogen concentration gradients have been proposed to control patterning as well as growth in many developing tissues. Some outstanding questions about tissue patterning by morphogen gradients are the following: What are the mechanisms that regulate gradient formation and shape? Is the positional information encoded in the gradient sufficiently precise to determine the positions of target gene domain boundaries? What are the temporal dynamics of gradients and how do they relate to patterning and growth? These questions are inherently quantitative in nature and addressing them requires measuring morphogen concentrations in cells, levels of downstream signaling activity, and kinetics of morphogen transport. Here we first present methods for quantifying morphogen gradient shape in which the measurements can be calibrated to reflect actual morphogen concentrations. We then discuss using fluorescence recovery after photobleaching to study the kinetics of morphogen transport at the tissue level. Finally, we present particle tracking as a method to study morphogen intracellular trafficking.

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

Systematic headgroup engineering yields planarizable push–pull flipper probes that are ready for use in biology – stable, accessible, modifiable –, and affords non-trivial insights into chalcogen-bond mediated mechanophore degradation and fluorescence enhancement.

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.

A multitude of biological processes that involve multiple interaction partners are observed by two-color microscopy. Here we describe an analysis method for the robust quantification of correlation between signals in different color channels: particle image cross-correlation spectroscopy (PICCS). The method, which exploits the superior positional accuracy obtained in single-object and single-molecule microscopy, can extract the correlation fraction and length scale. We applied PICCS to correlation measurements in living tissues. The morphogen Decapentaplegic (Dpp) was imaged in wing imaginal disks of fruit fly larvae and we quantified what fraction of early endosomes contained Dpp.

Tissue morphogenesis depends on the spatial arrangement of cells during development. A number of mechanisms have been described to contribute to the final shape of a tissue or organ, ranging from cell intercalation to the response of cells to chemotactic cues. One such mechanism is oriented cell division. Oriented cell division is determined by the position of the mitotic spindle. Indeed, there is increasing evidence implicating spindle misorientation in tissue and organ misshaping, which underlies disease conditions such as tumorigenesis or polycystic kidneys. Here we review recent studies addressing how the direction of tissue growth is determined by the orientation of cell division and how both extrinsic and intrinsic cues control the position of the mitotic spindle.

Sizing Signals Growth regulation of the Drosophila wing imaginal disc critically depends on the Decapentaplegic (Dpp) morphogen gradient. How a graded Dpp signal is interpreted by cells to control homogeneous tissue growth remains unclear. Wartlick et al. (p. 1154 ; see the Perspective by Le Goff and Lecuit ) address this question by measuring the spatial and temporal changes of Dpp concentration and signaling activity during the disc growth phase and by quantifying cell proliferation parameters in the discs. Both modeling and experimental findings suggest that both Dpp concentration and signaling gradients scale with tissue size so that, on average, the onset of mitosis occurs when Dpp signaling levels have increased by 50% since the beginning of the cell cycle.

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. In this study, we show that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, we demonstrate that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, our data demonstrate a fundamental role for a plasma membrane–endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration.

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.

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-β-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spaltexpression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

International audience; 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.

In this report, “fluorescent flippers” are introduced to create planarizable push–pull probes with the mechanosensitivity and fluorescence lifetime needed for practical use in biology. Twisted push–pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first “fluorescent flippers” are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns. Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET). These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy. Controls indicate that strong push–pull macrodipoles are important, operational probes do not relocate in response to lateral membrane reorganization, and two flippers are indeed needed to “really swim,” i.e., achieve high mechanosensitivity.

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR–Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging., Existing DNA stains for live cell microscopy are either toxic, require illumination with blue light, or are not compatible with super-resolution microscopy. Here the authors develop SiRHoechst, a non-toxic far-red DNA stain that is compatible with super-resolution microscopy.

During development, cells acquire positional information by reading the concentration of morphogens. In the developing fly wing, a gradient of the transforming growth factor–β (TGF-β)–type morphogen decapentaplegic (Dpp) is transduced into a gradient of concentration of the phosphorylated form of the R-Smad transcription factor Mad. The endosomal protein Sara (Smad anchor for receptor activation) recruits R-Smads for phosphorylation by the type I TGF-β receptor. We found that Sara, Dpp, and its type I receptor Thickveins were targeted to a subpopulation of apical endosomes in the developing wing epithelial cells. During mitosis, the Sara endosomes and the receptors therein associated with the spindle machinery to segregate into the two daughter cells. Daughter cells thereby inherited equal amounts of signaling molecules and thus retained the Dpp signaling levels of the mother cell.

The analysis of membrane trafficking has in the past mainly dealt with single cells in culture. Recent studies of membrane trafficking in Drosophila focus on how cells are organized in tissues and form epithelia during embryogenesis. During these processes, the specific involvement of distinct biosynthetic and endocytic routes is starting to be understood. Once organized in epithelia, cells communicate with each other to make cell fate decisions through morphogen gradients and lateral inhibition. Endocytosis seems to play unexpected roles in shaping morphogen gradients and in biasing lateral inhibition events. Once committed to a developmental program, cells differentiate. In the case of neurons, trafficking through the biosynthetic and endocytic pathways may give the necessary speed of response and versatility to axons that navigate through a changing environment during pathfinding.

The ability to internalize macromolecules by endocytosis is a property of all eukaryotic cells. Frontline research on endocytosis has been presented in a successful series of biannual meetings in Europe. This year's meeting on "Membrane Dynamics in Endocytosis" was held September 13-18 in Acquafredda di Maratea, on the coast of southern Italy. Four key questions were addressed: What are the molecular mechanisms of endocytic membrane trafficking? How does endocytosis modulate receptor signaling and vice versa? What is the importance of endocytosis during development? How do endocytic organelles contribute to immunity or susceptibility to pathogens?

Nowadays, the focus of developmental studies is shifting away from formal models of developmental pathways that are characterised by flow charts of controlling factors connected by arrows, to mechanistic models that explain developmental processes at the cellular level. Surprisingly, this shift towards a cellular view of developmental biology is occurring simultaneously across a range of model organisms. One consequence of taking such a cell biological view of development is that many model organisms are now becoming good models for studies of human disease and therapy.

During the last decade, many of the factors and mechanisms controlling membrane and protein trafficking in general and endocytic trafficking in particular have been uncovered. We have a detailed understanding of the different endocytic trafficking steps: plasma membrane budding, endocytic vesicle motility and fusion with the endosome, recycling, transcytosis and lysosomal degradation. The kinetics and trafficking pathway of many signaling receptors and the relevance of endocytic trafficking during signaling in many mammalian cultured cells are also well understood. However, only in recent years has the role of endocytic trafficking during cell-to-cell communication during development, i.e. during patterning, induction and lateral inhibition, begun to be explored. The contribution of Drosophila developmental genetics and cell biology has been fundamental in elucidating the essential role of endocytosis during these processes. Reviewed here are some of the recent developments on the role of endocytic trafficking during long- and short-range signaling and during lateral inhibition.

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division1,2. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila1. In flies, this has been generalized to stem cells in the gut3 and the central nervous system1, and, in zebrafish, to neural precursors of the spinal cord4. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

Secreted morphogens such as the Drosophila TGF-β homolog Decapentaplegic (Dpp) are thought to spread through target tissues and form long-range concentration gradients providing positional information. Using a GFP-Dpp fusion, we monitored a TGF-β family member trafficking in situ throughout the target tissue and forming a long-range concentration gradient. Evidence is presented that long-range Dpp movement involves Dpp receptor and Dynamin functions. We also show that the rates of endocytic trafficking and degradation determine Dpp signaling range. We propose a model where the gradient is formed via intracellular trafficking initiated by receptor-mediated endocytosis of the ligand in receiving cells with the gradient slope controlled by endocytic sorting of Dpp toward recycling versus degradation.

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor b (TGFb ) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concommittant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrinmediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells. q 1999 Elsevier Science Ireland Ltd. All rights reserved.

Three recent papers in Developmental Cell (by Yamamoto et al., Choi et al., and Sigismund et al.) show that the endocytic route of internalization can determine whether signaling receptors are degraded or recycled, and whether they initiate signaling from an endosomal platform. However, the same route can serve opposite functions in different signaling pathways: there is no "unifying theory" of trafficking.

International audience; Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GlcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.

NOTCH signaling is a developmental pathway known to play critical roles during embryonic development as well as for the regulation of self-renewing tissues. Aberrant activation of NOTCH signaling leads to deregulation of the self-renewal process resulting in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis, all of which are hallmarks of cancer. Over activation of NOTCH in human cancers can be a consequence of over expression of NOTCH ligands/receptors, GOF mutations in NOTCH receptors as well as chromosomal translocations leading to constitutive activation of the pathway. Given the importance of Notch signaling in human cancers, several therapeutic approaches have been utilized to block NOTCH signaling. Two of these strategies are; a) the use of monoclonal blocking antibodies (Mabs) against NOTCH ligands and receptors and b) the use of small molecule γ-secretase inhibitors (GSIs). However, these approaches can only be effective if tumor cells express full-length ligand or receptor molecules. On the contrary, in human cancers harbouring NOTCH gene fusion due to chromosomal translocations, the use of Mabs and GSIs will have very limited clinical benefits. A third, yet not fully explored approach could be the blockage of NOTCH signalling by targeting the most downstream event in the NOTCH cascade i,e NOTCH transcriptional activation complex using small molecule inhibitors. Here we report discovery and identification of a novel, orally-active small molecule inhibitor, named CB-103, of the NOTCH pathway that blocks NOTCH signaling by targeting the NOTCH transcriptional activation complex in the nucleus. CB-103 has shown the ability to block NOTCH signalling in human cancer cell lines, induce neurogenic phenotype in drosophila, induce muscle cell differentiation and inhibit NOTCH dependent cellular processes in mice. In addition, CB-103 exhibit anti-tumor efficacy in a xenograft model of human triple negative breast cancer resistant to GSIs and Mabs against NOTCH ligands/receptors. Furthermore, CB-103 has shown a remarkable activity in PDX models of human T-ALL harbouring activation of the NOTCH pathway. Additional studies are underway on several analogs of CB-103 to determine ADME/PK/PD profile and nominate a development candidate for further clinical development of this novel inhibitor of the NOTCH pathway. Citation Format: Rajwinder Lehal, Viktoras Frismantas, Sylvain Loubéry, Viktoria Reinmuller, Gerardo Turcatti, Marcos Gonzalez-Gaitan, Jean-Pierre Bourquin, Freddy Radtke. A novel small molecule inhibitor of the Notch transcription activation complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 338.

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 β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR 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.