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.     

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).     

Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization

Asymmetric localization is a prerequisite for inscuteable (insc) to function in coordinating and mediating asymmetric cell divisions in Drosophila. We show here that Partner of Inscuteable (Pins), a new component of asymmetric divisions, is required for Inscuteable to asymmetrically localize. In the absence of pins, Inscuteable becomes cytoplasmic and asymmetric divisions of neuroblasts and mitotic domain 9 cells show defects reminiscent of insc mutants. Pins colocalizes with Insc and interacts with the region necessary and sufficient for directing its asymmetric localization. Analyses of pins function in neuroblasts reveal two distinct steps for Insc apical cortical localization: A pins-independent, bazooka-dependent initiation step during delamination (interphase) and a later maintenance step during which Baz, Pins, and Insc localization are interdependent.     

Membrane targeting and asymmetric localization of Drosophila partner of inscuteable are discrete steps controlled by distinct regions of the protein

Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and G alpha i, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the G alpha i binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.     

Microtubule-induced Pins/Galphai cortical polarity in Drosophila neuroblasts

Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. We show that astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Galphai in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Galphai cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division.     

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.     

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.     

Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai; in addition, Gbeta13F is also required. However, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Galphai mutants and show that Galphai and Gbeta13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gbeta13F appears to regulate the asymmetric localization/stability of all apical components, and Gbeta13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.     

Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells

Asymmetric distribution of fate determinants is a fundamental mechanism underlying the acquisition of distinct cell fates during asymmetric division. In Drosophila neuroblasts, the apical DmPar6/DaPKC complex inhibits Lethal giant larvae (Lgl) to promote the basal localization of fate determinants. In contrast, in the sensory precursor (pI) cells that divide asymmetrically with a planar polarity, Lgl inhibits Notch signaling in the anterior pI daughter cell, pIIb, by a yet-unknown mechanism. We show here that Lgl promotes the cortical recruitment of Partner of Numb (Pon) and regulates the asymmetric distribution of the fate determinants Numb and Neuralized during the pI cell division. Analysis of Pon-GFP and Histone2B-mRFP distribution in two-color movies confirmed that Lgl regulates Pon localization. Moreover, posterior DaPKC restricts Lgl function to the anterior cortex at mitosis. Thus, Lgl functions similarly in neuroblasts and in pI cells. We also show that Lgl promotes the acquisition of the pIIb cell fate by inhibiting the plasma membrane localization of Sanpodo and thereby preventing the activation of Notch signaling in the anterior pI daughter cell. Thus, Lgl regulates cell fate by controlling Pon cortical localization, asymmetric localization of Numb and Neuralized, and plasma-membrane localization of Sandopo.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled remains unclear. Here we show that mitotic spindle geometry and unequal daughter cell size are controlled by two parallel pathways (Bazooka/DaPKC and Pins/G alpha i) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequal size neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/G alpha i localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle.     

Dlg1 controls planar spindle orientation in the neuroepithelium through direct interaction with LGN

Oriented cell divisions are necessary for the development of epithelial structures. Mitotic spindle orientation requires the precise localization of force generators at the cell cortex via the evolutionarily conserved LGN complex. However, polarity cues acting upstream of this complex in vivo in the vertebrate epithelia remain unknown. In this paper, we show that Dlg1 is localized at the basolateral cell cortex during mitosis and is necessary for planar spindle orientation in the chick neuroepithelium. Live imaging revealed that Dlg1 is required for directed spindle movements during metaphase. Mechanistically, we show that direct interaction between Dlg1 and LGN promotes cortical localization of the LGN complex. Furthermore, in human cells dividing on adhesive micropatterns, homogenously localized Dlg1 recruited LGN to the mitotic cortex and was also necessary for proper spindle orientation. We propose that Dlg1 acts primarily to recruit LGN to the cortex and that Dlg1 localization may additionally provide instructive cues for spindle orientation.     

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.     

Par-1 and PP2A: Yin-Yang of Bazooka Localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.     

Asymmetric localizations of LIN-17/Fz and MIG-5/Dsh are involved in the asymmetric B cell division in C. elegans

LIN-44/Wnt and LIN-17/Frizzled (Fz) function in a planar cell polarity (PCP)-like pathway to regulate the asymmetric B cell division in Caenorhabditis elegans. We observed asymmetric localization of LIN-17/Frizzled (Fz) and MIG-5/Dishevelled (Dsh) during the B cell division. LIN-17::GFP was asymmetrically localized within the B cell prior to and after the B cell division and correlated with B cell polarity. Asymmetric localization of LIN-17::GFP was dependent upon LIN-44/Wnt and MIG-5/Dsh function. The LIN-17 transmembrane domain and a portion of the cysteine-rich domain (CRD) were required for LIN-17 function and asymmetric distribution to the B cell daughters, while the conserved KTXXXW motif was only required for function. MIG-5::GFP was also asymmetrically localized within the B cell prior to and after the B cell division in a LIN-17- and LIN-44-dependent manner. Functions of the MIG-5 DEP, PDZ and DIX domains were also conserved. Thus, a novel PCP-like pathway, in which LIN-17 and MIG-5 are asymmetrically localized, is involved in the regulation of B cell polarity.     

The frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/beta-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz(-) patches reorient toward the fz(-) cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Our in vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, we demonstrate biochemical and physical interactions between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). We show that this function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. Our data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field.     

Par-1 and PP2A: Yin-Yang of Bazooka localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

Minimal amino acids in the I/LWEQ domain required for anterior/posterior localization in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Establishment of cell polarity is mediated by a series of signaling molecules that are asymmetrically activated or localized in the cell upon extracellular stimulation. To understand the mechanism that mediates anterior/posterior asymmetric localization of RapGAP3 during migration, we determined the minimally required amino acids in the I/LWEQ domain that cause posterior localization and found that the minimal region of the F-actin binding domain for posterior localization could, with some additional deletion at the C-terminal, localize to the anterior. Analysis of the localization and translocation kinetics to the cell cortex of the truncated proteins suggests that the required regions for anterior/posterior localization might have a preferential binding affinity to preexisting F-actins at the rear and lateral sides of the cell or newly formed F-actins at the front of the cell, leading to distinct differential sites of the cell.