Drosophila Sensory Organ Precursor Research Articles

Cell-intrinsic and -extrinsic functions of the ESCRT-III component shrub in cytokinetic abscission of Drosophila sensory organ precursor.

Although the molecular mechanisms governing abscission of isolated cells are largely decrypted, those 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 precursor (SOP). We report 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. SJs assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to a quicker disentanglement of neighboring cells membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions, 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.

Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila

The coordination between cell proliferation and cell polarity is crucial to orient the asymmetric cell divisions to generate cell diversity in epithelia. In many instances, the Frizzled/Dishevelled planar cell polarity pathway is involved in mitotic spindle orientation, but how this is spatially and temporally coordinated with cell cycle progression has remained elusive. Using Drosophila sensory organ precursor cells as a model system, we show that Cyclin A, the main Cyclin driving the transition to M-phase of the cell cycle, is recruited to the apical-posterior cortex in prophase by the Frizzled/Dishevelled complex. This cortically localized Cyclin A then regulates the orientation of the division by recruiting Mud, a homologue of NuMA, the well-known spindle-associated protein. The observed non-canonical subcellular localization of Cyclin A reveals this mitotic factor as a direct link between cell proliferation, cell polarity and spindle orientation.

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.

Notch Signaling: Where Is the Action?

It has been a long-standing question as to whether the activation of Notch by its ligands occurs in a specific region of the plasma membrane. A study now shows that this is indeed the case in the Drosophila sensory organ precursor cell lineage.

Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells

Notch receptors regulate cell fate decisions during embryogenesis and throughout adult life. In many cell lineages, binary fate decisions are mediated by directional Notch signaling between the two sister cells produced by cell division. How Notch signaling is restricted to sister cells after division to regulate intra-lineage decision is poorly understood. More generally, where ligand-dependent activation of Notch occurs at the cell surface is not known, as methods to detect receptor activation invivo are lacking. In Drosophila pupae, Notch signals during cytokinesis to regulate the intra-lineage pIIa/pIIb decision in the sensory organ lineage. Here, we identify two pools of Notch along the pIIa-pIIb interface, apical and basal to the midbody. Analysis of the dynamics of Notch, Delta, and Neuralized distribution in living pupae suggests that ligand endocytosis and receptor activation occur basal to the midbody. Using selective photo-bleaching of GFP-tagged Notch and photo-tracking of photo-convertible Notch, we show that nuclear Notch is indeed produced by receptors located basal to the midbody. Thus, only a specific subset of receptors, located basal to the midbody, contributes to signaling in pIIa. This is the first invivo characterization of the pool of Notch contributing to signaling. We propose a simple mechanism of cell fate decision based onintra-lineage signaling: ligands and receptors localize during cytokinesis to the new cell-cell interface, thereby ensuring signaling between sister cells, hence intra-lineage fate decision.

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division.

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. We show that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery.

Planar Cell Polarity breaks the symmetry of PAR protein distribution prior to mitosis in Drosophila Sensory Organ Precursor cells

During development, cell fate diversity can result from the unequal segregation of fate determinants at mitosis [1]. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6 and atypical Protein Kinase C (aPKC) [1-5]. In the fly notum, Sensory Organ Precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells [6-12]_ENREF_12. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of Planar Cell Polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis [6, 13-15]. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6 and aPKC in living pupae. Using imaging of functional GFP-tagged proteins with image processing and computational modelling, we find that Baz, Par6 and aPKC become planar polarized prior to mitosis in a manner independent of the Aurora-A kinase and that PCP is required for the planar polarization of Baz, Par6 and aPKC during interphase. This indicates that a ‘mitosis rescue’ mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs.

The Conserved Discs-large Binding Partner Banderuola Regulates Asymmetric Cell Division in Drosophila

Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, we have carried out an invivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.

Regulation of centrosome movements by Numb and the Collapsin Response Mediator Protein during Drosophila sensory progenitor asymmetric division

Asymmetric cell division generates cell fate diversity during development and adult life. Recent findings have demonstrated that during stem cell divisions, the movement of centrosomes is asymmetric in prophase and that such asymmetry participates in mitotic spindle orientation and cell polarization. Here, we have investigated the dynamics of centrosomes during Drosophila sensory organ precursor asymmetric divisions and find that centrosome movements are asymmetric during cytokinesis. We demonstrate that centrosome movements are controlled by the cell fate determinant Numb, which does not act via its classical effectors, Sanpodo and α-Adaptin, but via the Collapsin Response Mediator Protein (CRMP). Furthermore, we find that CRMP is necessary for efficient Notch signalling and that it regulates the duration of the pericentriolar accumulation of Rab11-positive endosomes, through which the Notch ligand, Delta is recycled. Our work characterizes an additional mode of asymmetric centrosome movement during asymmetric divisions and suggests a model whereby the asymmetry in centrosome movements participates in differential Notch activation to regulate cell fate specification.

Notch Signaling: Numb Makes the Difference

During Drosophila sensory organ precursor cell development, Numb segregates asymmetrically and functions as a cell fate determinant. Recent work now demonstrates in vivo that Numb inactivates Notch by promoting its endocytosis.

Numb and Alzheimer’s Disease: The Current Picture

Twenty-three years ago, numb was identified as a critical regulator in Drosophila sensory organ precursor cell asymmetric divisions. Beyond the recently recognized role in carcinogenesis, Numb seems to be important in Alzheimer’s disease. This assertion comes from the involvement in various processes such as synapse morphogenesis, amyloid precursor protein trafficking, notch signaling, and neurogenesis. The purpose of the present mini-review is to provide the current picture of Numb’s participation in mechanisms underlying Alzheimer’s disease pathogenesis and emphasize potential aspects for future research.

Numb Independently Antagonizes Sanpodo Membrane Targeting and Notch Signaling in Drosophila Sensory Organ Precursor Cells

In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure-function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs.

Targeting of Sanpodo to asymmetric pericentrosomal early endosomes regulates Notch signaling in Drosophila sensory organ precursor cells

Targeting of Sanpodo to asymmetric pericentrosomal early endosomes regulates Notch signaling in Drosophila sensory organ precursor cells

Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila.

During asymmetric division, a cell polarizes and differentially distributes components to its opposite ends. The subsequent division differentially segregates the two component pools to the daughters, which thereby inherit different developmental directives. In Drosophila sensory organ precursor cells, the localization of Numb protein to the cell's anterior cortex is a key patterning event and is achieved by the combined action of many proteins, including Pins, which itself is localized anteriorly. Here, a role is described for the trimeric G protein Go in the anterior localization of Numb and daughter cell fate specification. Go is shown to interact with Pins. In addition to a role in recruiting Numb to an asymmetric location in the cell's cortex, Go transduces a signal from the Frizzled receptor that directs the position in which the complex forms. Thus, Go likely integrates the signaling that directs the formation of the complex with the signaling that directs where the complex forms.

Senseless physically interacts with proneural proteins and functions as a transcriptional co-activator

The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop-helix (bHLH) proneural proteins in Drosophila sensory organ precursors and is required for their normal development. High levels of Senseless synergize with bHLH proteins and upregulate target gene expression, whereas low levels of Senseless act as a repressor in vivo. However, the molecular mechanism for this dual role is unknown. Here, we show that Senseless binds bHLH proneural proteins via its core zinc fingers and is recruited by proneural proteins to their target enhancers to function as a co-activator. Some point mutations in the Senseless zinc-finger region abolish its DNA-binding ability but partially spare the ability of Senseless to synergize with proneural proteins and to induce sensory organ formation in vivo. Therefore, we propose that the structural basis for the switch between the repressor and co-activator functions of Senseless is the ability of its core zinc fingers to interact physically with both DNA and bHLH proneural proteins. As Senseless zinc fingers are approximately 90% identical to the corresponding zinc fingers of its vertebrate homologue Gfi1, which is thought to cooperate with bHLH proteins in several contexts, the Senseless/bHLH interaction might be evolutionarily conserved.

Quantitative Analysis of Protein Dynamics during Asymmetric Cell Division

In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis.

Asymmetric Rab11 Endosomes Regulate Delta Recycling and Specify Cell Fate in the Drosophila Nervous System

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.

Numb and α‐Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.

Sec15, a Component of the Exocyst, Promotes Notch Signaling during the Asymmetric Division of Drosophila Sensory Organ Precursors

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, we have isolated a mutation in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15(-) sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, we propose that a defect in Delta recycling causes cell fate transformation in sec15(-) sensory lineages. Our data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila.