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.     

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.     

Overexpression of partner of numb induces asymmetric distribution of the PI4P 5-Kinase Skittles in mitotic sensory organ precursor cells in Drosophila

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::Pon(LD)) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::Pon(LD) is often used as a polarity marker in dividing progenitors.     

Regulation of membrane localization of Sanpodo by lethal giant larvae and neuralized in asymmetrically dividing cells of Drosophila sensory organs

In Drosophila, asymmetric division occurs during proliferation of neural precursors of the central and peripheral nervous system (PNS), where a membrane-associated protein, Numb, is asymmetrically localized during cell division and is segregated to one of the two daughter cells (the pIIb cell) after mitosis. numb has been shown genetically to function as an antagonist of Notch signaling and also as a negative regulator of the membrane localization of Sanpodo, a four-pass transmembrane protein required for Notch signaling during asymmetric cell division in the CNS. Previously, we identified lethal giant larvae (lgl) as a gene required for numb-mediated inhibition of Notch in the adult PNS. In this study we show that Sanpodo is expressed in asymmetrically dividing precursor cells of the PNS and that Sanpodo internalization in the pIIb cell is dependent cytoskeletally associated Lgl. Lgl specifically regulates internalization of Sanpodo, likely through endocytosis, but is not required for the endocytosis Delta, which is a required step in the Notch-mediated cell fate decision during asymmetric cell division. Conversely, the E3 ubiquitin ligase neuralized is required for both Delta endocytosis and the internalization of Sanpodo. This study identifies a hitherto unreported role for Lgl as a regulator of Sanpodo during asymmetric cell division in the adult PNS.     

Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.     

Asymmetric Rab 11 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.     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.     

Lethal giant larvae acts together with numb in notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage

The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. We report here that, in contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages.     

Numb and alpha-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.     

Phosphorylation-induced autoinhibition regulates the cytoskeletal protein Lethal (2) giant larvae

During asymmetric cell division, cell fate determinants localize asymmetrically and segregate into one of the two daughter cells. In Drosophila neuroblasts, the asymmetric localization of cell fate determinants to the basal cell cortex requires aPKC. aPKC localizes to the apical cell cortex and phosphorylates the cytoskeletal protein Lethal (2) giant larvae (Lgl). Upon phosphorylation, Lgl dissociates from the cytoskeleton and becomes inactive. Here, we show that phosphorylation regulates Lgl by allowing an autoinhibitory interaction of the N terminus with the C terminus of the protein. We demonstrate that interaction with the cytoskeleton is mediated by a C-terminal domain while the N terminus is not required. Instead, the N terminus can bind to the C terminus and can compete for binding to the cytoskeleton. Interaction between the N- and C-terminal domains requires phosphorylation of Lgl by aPKC. Our results suggest that unphosphorylated, active Lgl exists in an open conformation that interacts with the cytoskeleton while phosphorylation changes the protein to an autoinhibited state.     

Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage

The Drosophila bristle lineage is an excellent system in which to study how cell cycle and fate determination are synchronized in invariant cell lineages. In this model, five different cells arise from a single precursor cell, pI, after four asymmetric cell divisions. Cell diversity is achieved by the asymmetric segregation of cell determinants, such as Numb and Neuralized (Neur), resulting in differential activation of the Notch (N) pathway. We show that down-regulation of Cdc2, by over-expressing Tribbles, Dwee1, and Dmyt1 (three negative regulators of Cdc2) or by using thermo-sensitive Cdc2 mutant flies, delayed pI mitosis, and altered the polarity and the number of subsequent cell divisions. These modifications were associated with a mother-daughter cell fate transformation as the pI cell acquired the identity of the secondary precursor cell, pIIb. This type of change in cell identity only occurred when the N signaling pathway was inactive since ectopic N signaling transformed pI to pIIa-progeny fate. These transformations in cell identity suggest that, although synchronized, cell cycle and fate determination are independent phenomena in the bristle lineage.     

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.     

Dlg,Scrib and Lgl regulate neuroblast cell size and mitotic spindle asymmetry

Asymmetric cell division is important in generating cell diversity from bacteria to mammals. Drosophila melanogaster neuroblasts are a useful model system for investigating asymmetric cell division because they establish distinct apical-basal cortical domains, have an asymmetric mitotic spindle aligned along the apical-basal axis, and divide unequally to produce a large apical neuroblast and a small basal daughter cell (GMC). Here we show that Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) tumour suppressor proteins regulate multiple aspects of neuroblast asymmetric cell division. Dlg/Scrib/Lgl proteins show apical cortical enrichment at prophase/metaphase, and then have a uniform cortical distribution. Mutants have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size. Defects in apical cell and spindle pole size result in symmetric or inverted neuroblast cell divisions. Inverted divisions correlate with the appearance of abnormally small neuroblasts and large GMCs, showing that neuroblast/GMC identity is more tightly linked to cortical determinants than cell size. We conclude that Dlg/Scrib/Lgl are important in regulating cortical polarity, cell size asymmetry and mitotic spindle asymmetry in Drosophila neuroblasts.     

The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.     

Drosophila E-cadherin regulates the orientation of asymmetric cell division in the sensory organ lineage

BACKGROUND: Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the one of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. RESULTS: DE-Cadherin-Catenin complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized DE-Cadherin-Catenin complexes. While a complete loss of DE-Cadherin function disrupts the apical-basal polarity of the epithelium, both a partial loss of DE-Cadherin function and expression of a dominant-negative form of DE-Cadherin affect the orientation of the pIIa division. Furthermore, expression of dominant-negative DE-Cadherin also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Cad regulates the orientation of asymmetric cell division. CONCLUSIONS: We describe a novel mechanism involving a specialized Cad-containing cortical region by which a daughter cell divides with the same orientation as its mother cell.     

The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila

During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch.     

Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.