In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumor-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).
In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch signaling, associate with their respective adapter proteins, Miranda and Partner of Numb (Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz) and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of the basal determinants. However, the mechanisms that underlie the asymmetric protein sorting in neuroblasts are not known. To address this issue, chromosomal deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the basal cortex upon mitosis after a transient spread into the cytoplasm. In germline clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule structures. Consequently, Miranda segregates into both the daughter neuroblast and the ganglion mother cell (GMC). Numb and Pon are also distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).
Lgl is involved in epithelial polarity, and neuroblasts inherit Baz as an apical polarity cue during the formation from neuroepithelia. In epithelia lacking lgl function, Baz mislocalizes as irregular patches. However, Baz as well as Insc and Pins normally localize as an apical crescent in the mutant neuroblasts, suggesting that the apical cue is inherited by neuroblasts in the absence of lgl. Moreover, neuroblast-specific expression of the lgl transgene can restore normal localization of Miranda. Thus, Lgl probably functions autonomously within neuroblasts for Miranda localization, and the phenotype in lgl neuroblasts would not result from the inheritance of an abnormal polarity cue. Together, it is concluded that Lgl acts in the localization of the basal determinants in neuroblasts, but not of the apical Baz-Insc-Pins complex (Ohshiro, 2000).
Whether other tumor-suppressor genes contribute to protein localization in neuroblasts was investigated. The tumor-suppressor gene dlg encodes a membrane-associated guanylate kinase homolog. Germline clone embryos lacking both maternal and zygotic dlg activity (dlgGLC embryos) exhibit defective localization of Miranda and Numb essentially identical to that of lglGLC embryos, suggesting that both tumor-suppressor proteins function in the same process in neuroblasts. To investigate the relationship between the roles of Dlg and Lgl, their subcellular localization in neuroblasts was compared. Both Lgl and Dlg are distributed mainly throughout the cortex, whereas the amount of Lgl in the cytoplasm appears to be greater in mitosis than in interphase. The cortical localization of Lgl appears to be important for its function -- whereas the mutant protein encoded by the temperature-sensitive allele lglts3 is distributed normally at the permissive temperature (18°C), it fails to localize cortically at the restrictive temperature (29°C). The wild-type Lgl protein exhibits a similar, abnormal cytoplasmic distribution in dlgGLC embryos, whereas Dlg localization is not affected in lglGLC embryos. Thus, the cortical localization of Lgl requires dlg activity, suggesting that Dlg may function in localization of cell-fate determinants in neuroblasts by positioning Lgl at the cortex (Ohshiro, 2000).
To distinguish whether Lgl establishes or maintains determinant localization, temperature-shift experiments were performed with the lglts3 allele. At 18°C, most (97%) lgltsc/lgl minus embryos of females homozygous for lgltsc exhibit normal Miranda localization at the basal cortex during metaphase and telophase. Ten minutes after shifting to 29°C, mislocalization of Miranda is apparent in 61% of metaphase or anaphase neuroblasts as well as in 43% of telophase neuroblasts, whereas the apical localization of Miranda in interphase is not affected. On the basis of live recordings of basal crescent formation in cells that express a fusion protein comprising Miranda and green fluorescent protein, it is estimated that the time required for neuroblast mitosis is 14.3+/-2.4 min. Thus, a temperature shift during mitosis is able to induce mislocalization of Miranda, as is apparent in telophase neuroblasts, indicating that Lgl functions during mitosis to determine Miranda localization. Metaphase arrest can be induced in lglts3/lgl minus embryos by introduction of the fizzy (fzy) mutation. The localization of Miranda in such metaphase-arrested neuroblasts is virtually insensitive to the shift to 29°C. Thus, Miranda is not affected by the decrease in lgl function after it has localized to the basal cortex. These results therefore indicate that Lgl may act early during mitosis to recruit Miranda to the cortex but does not contribute to the maintenance of Miranda in this location (Ohshiro, 2000).
Because Lgl is a component of cortical protein complexes that include nonmuscle Myosin II, or Zipper (Zip), a test was performed for genetic interactions between lgl and zip in Miranda localization by examining embryos zygotically mutant for both lgl and zip. The zip1 mutation does not affect Miranda localization throughout embryonic development and lgl-zip embryos show no difference in Miranda localization from zygotic lgl- embryos until late embryonic stages (stage 16) owing to the maternal contribution of zip. However, at stage 17 when maternal zip had been exhausted, lgl-zip embryos appear to restore the basal crescent of Miranda in metaphase neuroblasts, whereas zygotic lgl- embryos at the same stage do not. Thus, Lgl might act for Miranda localization in part by suppressing zip function directly or indirectly, consistent with a study on yeast that indicated negative genetic interactions between Lgl homologs and Myosin II. Alternatively, the asymmetric distribution of Pon requires myosin function in neuroblasts, as revealed by the use of 2,3-butanedione monoxime (BDM) that generally inhibits myosin function. The effect of BDM on Miranda localization was examined. Treatment of wild-type embryos with BDM phenocopies lgl mutants, resulting in a partial redistribution of Miranda from the cortex to microtubules. The effect of BDM is more marked in lglGLC embryos: as the BDM concentration increases, the relocalization of Miranda to microtubules is synergistically enhanced in most BDM-treated neuroblasts and results in the complete exclusion of Miranda from the cortex at 50 mM BDM. The phenocopy and enhancement of lgl mutations by general inhibition of myosin function are in contrast with the suppressive effects of zip mutations, suggesting that Lgl cooperates with at least one type of Myosin other than Zip to anchor Miranda at the cell cortex. It is thus inferred that Lgl regulates negatively myosin II function and also positively the function of another Myosin isotype in cortical protein targeting in neuroblasts (Ohshiro, 2000).
It would be expected that the abnormal distribution of Numb and Miranda in lgl mutant neuroblasts results in incorrect determination of neural cell fate. Given the difficulty of monitoring neural cell fate in severely distorted lglGLC embryos, this prediction was tested by analyzing the lineage of the external sensory organ in the notum, in which all cell divisions are asymmetric and sibling cells adopt distinct fates as a result of the asymmetric inheritance of Numb. Sensory organ precursor cells in this lineage segregate Numb into a daughter cell pIIb, which subsequently generates three inner cells (a glial cell, a neuron and a sheath cell). The sibling pIIa cell divides into two outer cells constituting the external sensory structure, a hair and a socket. Exposure of lglts3 mutant larvae to 29°C during external sensory organ development mislocalizes Numb in mitotic precursor cells, as observed in neuroblasts, and often transforms inner cells into outer cells resulting in duplicated external sensory structures, a phenotype expected from loss of numb function. Indeed, this notum phenotype is enhanced by reducing the numb gene dosage by half. Equal partition of Numb between sibling cells would result in numb gain of function phenotypes because the half dose of numb is enough for correct cell-fate decisions. The observed numb loss-of-function phenotype therefore suggests that a reduction in lgl activity does not only equalize Numb distribution between sibling cells but also attenuates numb function, consistent with the observation of cytoplasmic Numb in the lgl mutants. Conversely, the presence of an extra numb gene induces opposite phenotypes under the lgl mutant condition. The outer cells are frequently transformed into the inner cells, resulting in the loss of the external sensory structure. This appearance of the numb gain of function phenotypes is simply explained by the fact that the partition of additional Numb from the transgene into both sibling cells raises numb activities over the threshold necessary to suppress Notch function in both cells. These data thus indicate that Lgl is essential in neural fate decisions through cortical targeting of cell-fate determinants (Ohshiro, 2000).
There are two important processes associated with the asymmetric division: (1) the asymmetric localization of cell-fate determinants, which is achieved by specific adapter proteins that themselves localize asymmetrically to the cortex in neuroblasts; and (2) the orientation of the mitotic spindle and its coordination with the polarized localization of the determinants, which requires the apical Baz-Insc-Pins complex. This study has revealed another important process mediated by Dlg, Lgl and Myosins, which is responsible for the cortical anchoring of the determinant-adapter complexes. This process occurs upstream of the first and independently or parallel to the second of those two aspects of asymmetric division, as the localization of Lgl and Dlg is independent of apical or basal components. Both Lgl and Dlg contribute to the generation or maintenance of epithelial polarity, and zygotic mutants of the corresponding genes develop epithelial cell tumors as well as brain tumors at late larval stages. These previous observations with epithelial cells, together with the data on the roles of Lgl and Dlg in protein targeting in neuroblasts, suggest that aberrant sorting of intracellular proteins may be responsible for the tumor formation apparent in larval stages of lgl and dlg mutants (Ohshiro, 2000).
Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).
Embryonic Drosophila neuroblasts develop from an apical/basal polarized epithelium. Individual cells delaminate into the embryo, enlarge to form neuroblasts, and begin a series of asymmetric cell divisions; these divisions result in the production of a large mitotically active apical cell (neuroblast), and a smaller basal cell (ganglion mother cell, GMC) that differentiates into two neurons or glia. A growing number of proteins are known to be asymmetrically localized in mitotic neuroblasts: apically localized proteins include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins); basally targeted proteins include Miranda, Prospero, Partner of Numb (Pon) and Numb, which are important for GMC development. Miranda and Prospero are apically localized at late interphase before their mitosis-dependent transport to the basal cortex. The Baz/Insc/Pins apical complex is required for both apical/basal spindle orientation and basal protein targeting, but little is known about how this complex regulates either process (Peng, 2000).
To identify genes required for apical/basal protein targeting in neuroblasts, deficiency stocks were screened looking for defects in Prospero basal localization in neuroblasts. This screen identified the lgl gene, which encodes a WD-40 repeat protein with homologues in many species, including the closely related 'Lgl family' genes Lgl1/Lgl2 (human), Lgl1 (mouse), U51993 (Caenorhabditis elegans); the slightly more divergent 'Tomosyn family' genes Tomosyn (rat), KIAA1006 (human), Tomosyn (Drosophila), and M01A10 (C. elegans); and recently duplicated genes similar to both families: sro7/sro77 (budding yeast). In Drosophila, lgl mutations affect protein targeting to epithelial apical junctions, epidermal cell-shape changes, and produce tumors of the brain and the imaginal disc. This spectrum of phenotypes has been noted for another tumor-suppressor gene, discs large. This study explores the role of Lgl and Dlg in regulating neuroblast cell polarity (Peng, 2000).
Apical and basal protein targeting are compared in neuroblasts from wild-type embryos and embryos that lack all maternal and zygotic Lgl or Dlg function (called lglGLC or dlgGLC embryos). Wild-type metaphase neuroblasts show apical Insc/Pins localization, and basal Miranda/Prospero/Pon crescents. In addition, Miranda and Prospero proteins can be observed around the apical centrosome and weakly on the mitotic spindle in wild-type neuroblasts. In contrast, all lglGLC and dlgGLC metaphase neuroblasts show cytoplasmic Pon and uniformly cortical and strongly spindle-associated Miranda/Prospero; the apical proteins Insc/Pins are normal or slightly expanded. Although lglGLC and dlgGLC embryos show striking defects in neuroblast basal protein localization, they also show an early loss of embryonic epithelial apical/basal polarity, which could indirectly cause the observed neuroblast defects (Peng, 2000).
To determine the neuroblast-specific function of Lgl and Dlg, Lgl- or Dlg-depleted neuroblasts were studied in embryos or larvae where epithelial development occurs normally. Initially, homozygous null lgl4 embryos were studied, in which maternal Lgl protein allows normal embryonic epithelial development (including Armadillo, Crumbs and Dlg localization). In stage 16-17 lgl4 embryos, mitotic neuroblasts show normal Baz/Insc/Pins apical crescents, and normal spindle orientation, but Miranda/Prospero are delocalized onto the spindle and around the cortex and Pon is cytoplasmic. This phenotype is less severe in early embryos but fully penetrant in older embryos, presumably due to progressive loss of maternal Lgl protein. Next, neuroblasts were assayed in lgl3344 or dlgv55 homozygous larvae -- these larval neuroblasts are persistent embryonic neuroblasts that develop from a normal embryonic epithelium due to maternal Lgl and Dlg protein function. Wild-type larval metaphase neuroblasts have Insc/Pins crescents the opposite of Miranda/Prospero/Pon/Numb crescents, whereas homozygous lgl3344 or dlgv55 larval metaphase neuroblasts show normal Insc/Pins crescents but Miranda/Prospero/Pon proteins are cytoplasmic, uniformly cortical, and weakly spindle-associated. It is concluded that Lgl and Dlg are required specifically in neuroblasts for basal protein targeting, without affecting apical protein localization or spindle orientation (Peng, 2000).
The lgl and dlg neuroblast basal localization phenotype is cell-cycle dependent. lgl and dlg mutant embryonic and larval neuroblasts show a fully penetrant loss of basal protein targeting at metaphase, but by late anaphase or telophase most neuroblasts show normal basal protein localization; 'telophase rescue' of basal protein localization also occurs in baz, insc and pon mutants. These results indicate that there are probably multiple mechanisms for basal protein localization (Peng, 2000).
Delocalization of the Prospero and Numb proteins produces defects in the nervous system and other tissues, so lgl mutant embryos were scored for cell fate defects. lglGLC embryos have severe morphological defects that preclude analysis, and lgl4 embryos can only be scored for late embryonic phenotypes, due to persistence of maternal Lgl protein. lgl4 embryos show a decrease in Even-skipped lateral (EL) neuron number at stage 17. A similar but stronger phenotype is seen in numb mutants, suggesting that the lgl phenotype may be due to delocalization of Numb during the GMC divisions that produce the EL neurons. The relatively mild lgl phenotype could be due to 'telophase rescue' of Numb protein in these GMCs, or to maternal Lgl protein (Peng, 2000).
In wild-type embryonic neuroblasts, Dlg protein is cortical with an apical crescent from late interphase to the end of mitosis that co-localizes with Baz/Insc/Pins, whereas Lgl protein is uniformly cortical and weakly cytoplasmic. Similarly, larval neuroblasts show apical Dlg and uniform cortical/cytoplasmic Lgl localization. lgl mutants show normal Dlg localization, but dlg mutants show loss of cortical Lgl protein. Thus, Dlg acts upstream of Lgl for its localization, but not necessarily its function (Peng, 2000).
How does Lgl regulate basal protein targeting? Lgl binds non-muscle myosin II in all organisms tested, and sro7/77 and myo1 (encoding Lgl-related proteins and myosin II, respectively) show strong negative genetic interactions in yeast. Tests were performed for genetic interactions between lgl4 and two different null mutations in zipper (encoding myosin II), scoring Miranda basal localization in stage 17 neuroblasts, when maternal Lgl and Myosin II protein levels are lowest. Wild-type and zip embryos have normal basal protein localization, whereas lgl4 embryos show complete delocalization of basal proteins. However, lgl4 embryos lacking one copy of myosin II show a significant increase in basal protein targeting; and lgl4;zip1 mutant embryos show virtually normal basal protein targeting. Thus, reducing myosin II levels strongly suppresses the lgl phenotype, indicating that myosin II can inhibit basal targeting when Lgl levels are low (Peng, 2000).
In addition, the general myosin inhibitor 2,3-butanedione monoxime (BDM) can suppress the lgl phenotype: stage 10 lgl4 embryos treated with BDM show a significant increase in basal protein localization compared with sham-treated stage 10 lgl4 embryos. Wild-type or lgl4 embryos treated with 50 mM BDM show delocalization of Miranda, Prospero and Pon. These data indicate that a myosin that is sensitive to 25 mM BDM inhibits basal protein localization in lgl embryos (probably myosin II), and at least one myosin that is sensitive to 50 mM BDM promotes basal protein targeting in mitotic neuroblasts (Peng, 2000).
Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).
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) following 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 central nervous system (CNS). lethal giant larvae (lgl) is required for Numb-mediated inhibition of Notch in the adult PNS. Sanpodo is expressed in asymmetrically dividing precursor cells of the PNS and Sanpodo internalization in the pIIb cell is dependent on 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 (Roegiers, 2005).
This analysis of Sanpodo function in the adult PNS suggests that, as in the embryo, Sanpodo is expressed only in asymmetrically dividing precursor cells and is required for cell fates dependant on high levels of Notch signaling, perhaps through the direct interaction between Sanpodo and the full length Notch receptor. Sanpodo also interacts directly with Numb in vivo, and in both the embryonic CNS and the adult PNS, numb inhibits plasma membrane association of Sanpodo. Therefore, it appears that Sanpodo plays a similar role in asymmetrically dividing precursor cells in both the CNS and PNS in Drosophila (Roegiers, 2005).
Although there are many similarities between the mechanisms of asymmetric cell divisions in embryonic neuroblasts and adult sensory organ precursor cells, one difference involves the role of lgl. In neuroblasts, lgl is required along with another cortical tumor suppressor, dlg, to target Numb to a basal crescent during mitosis, whereas in pI cells, only dlg is required for Numb crescent formation. While lgl is dispensable for segregation of Numb to the pIIb cell following pI cell mitosis, lgl is required for the inhibition of Notch signaling in the pIIb cell. Based on the current study, it is proposed that Lgl functions with Numb to remove Sanpodo from the membrane, leading to down regulation of the Notch signaling pathway in the pIIb cell. Through what mechanism might Lgl regulate Sanpodo localization? Studies in Drosophila, yeast, and vertebrate cells have implicated Lgl as both a regulator of exocytosis, through its interaction with t-SNARES, and as cytoskeletal effector. In this study, no phenotypes suggesting gross defects in exocytosis were detected; in fact, increased accumulation of the membrane protein Sanpodo at the plasma membrane is seen in lgl mutants. Accumulation of Sanpodo at the plasma membrane in lgl mutants resembles the phenotype of three endocytic proteins Numb, alpha-Adaptin, and Shibire, suggested that lgl may have a broader role in vesicle traffic. Although a potential role for Lgl in endocytosis is observed, this role appears to be specific to Sanpodo, since endocytosis of Delta occurs normally in lgl mutants, suggesting that Lgl is not required for bulk endocytosis. Increasingly, selective endocytosis is being implicated as an important regulator of signaling pathways. Two recent studies demonstrate that Liquid facets, an endocytic epsin participates in the Neuralized-mediated Delta endocytosis, apparently by targeting mono-ubiquitinated Delta to a specific, activating, endocytic compartment. The Notch receptor is also subjected to an ubiquitin-mediated endocytic step required for activation via the E3 ubiquitin ligase Deltex, which targets Notch to the late endosome. However, the roles of Liquid facets and Deltex have not been explored in asymmetrically dividing neural precursors. One possible function for Lgl could be to direct Sanpodo toward a specific endocytic compartment. Alternatively, Lgl may be involved indirectly, by targeting molecules required for Sanpodo endocytosis to the membrane region. This scenario would be more consistent with Lgl's role as an exocytic regulator. An alternative hypothesis may be that Lgl regulates Sanpodo localization through its interaction with the cytoskeleton. Lgl functions as an inhibitor of non-muscle myosin II function in both Drosophila and yeast. The data suggests that cytoskeletal association of Lgl is required for regulating Sanpodo localization, because phosphorylation of Lgl by aPKC, which causes an autoinhibitory conformational change in Lgl that disrupts the association with the cytoskeleton, causes membrane accumulation of Sanpodo. It remains to be determined if Sanpodo endocytosis requires inhibition of myosin II activity (Roegiers, 2005).
Previously, Numb and Neuralized had been implicated in two complementary, and possibly independent, mechanisms to determine cell fate in PNS precursor cells. Numb functions to inhibit Notch autonomously by internalizing Sanpodo in the pIIb cell: while Neuralized acts on Delta in the pIIb cell to induce Notch signaling non-autonomously in the pIIa cell. Both neuralized-dependant uptake of Delta and Sanpodo internalization require dynamin function, suggesting that these steps rely on endocytosis. Unexpectedly, it was found that loss of neuralized function affects both Delta internalization and Sanpodo internalization. Failure to internalize Delta into the pIIb cell causes a cell fate transformation of the pIIa cell into a pIIb cell in neuralized mutants, and this transformation occurs despite the accumulation of Sanpodo at the membrane, suggesting that accumulation of Sanpodo at the membrane is not sufficient to induce Notch signaling in the pIIb cell in the absence of neuralized. It is unclear whether membrane accumulation of Sanpodo in neuralized mutants is due to a direct interaction between Neuralized and Sanpodo, perhaps through ubiquitination of Sanpodo, or through an indirect mechanism. Regardless, the data show that regulation of Sanpodo membrane localization is not completely independent of neuralized function. In summary, this study suggests that Sanpodo is regulated by both neuralized and lgl, while Delta is regulated by neuralized independently of lgl. In addition, this study shows that lgl appears to contribute to the endocytosis of Sanpodo, which suggests a broader role for lgl in vesicle trafficking, which may have important implications for its role as a tumor suppressor. Could the regulation of Notch signaling by Sanpodo, Lgl and Numb be conserved across species? Sequence analysis did not reveal any homologues of Sanpodo beyond other insect species. However, loss of function studies of the mouse homologues of Drosophila numb and lgl in the developing brain show strikingly similar phenotypes. Targeted numb/numblike knockouts in dorsal forebrain and Lgl1 knockouts cause profound disorganization of the layered regions of the cortex and striatum and formation of rosettelike accumulations of neurons. These phenotypes may indicate that Numb and Lgl function together to regulate Notch signaling in mouse neurogenesis as well as in Drosophila PNS development, but a functional homologue sanpodo has yet to be identified in the mouse (Roegiers, 2005).
Inactivation of the Drosophila lethal(2)giant larvae gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins the structure of the cytosolic form of p127 has been analyzed. Using gel filtration and immunoaffinity chromatography it has been found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicate that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, shows that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, one of the proteins has been identified by microsequencing as nonmuscle myosin II heavy chain. Blot overlay assay shows that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network (Strand, 1994b).
The p127 tumor suppressor protein encoded by the lethal(2)giant larvae gene is a component of a cytoskeletal network distributed in both the cytoplasm and on the inner face of the plasma membrane. The p127 protein forms high molecular mass complexes consisting mainly of homo-oligomerized p127 molecules and at least ten additional proteins. One of these proteins has been recently identified as nonmuscle myosin type II heavy chain. To determine the functional interactions between p127 and other proteins present in the p127 complexes, p127 was examined for posttranslational modifications. It was found that p127 can be phosphorylated at serine residues. In this report the characteristics of a serine kinase that is associated with p127, is described, as judged by its recovery in p127 complexes purified by either gel filtration or immuno-affinity chromatography. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127 and containing five serine residues that are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane (Kalmes, 1996).
The p127 tumor suppressor protein encoded by the l(2)gl forms high molecular mass complexes consisting predominantly of p127 molecules. To determine whether p127 can self-assemble in the absence of other binding factors, the size of in vitro synthesized p127 was analyzed by gel filtration and it was found that p127 is always recovered in a high molecular mass form, demonstrating that p127 can oligomerize on its own. Previous studies have revealed that p127 may contain three homo-oligomerization domains. To more accurately delineate these domains, a series of 32 chimaeric proteins has been generated made of defined portions of p127 fused to protein A, which behaves as a monomeric protein, and the level of oligomerization of the fused proteins has been determined. This study allowed the mapping of three discrete homo-oligomerization domains, each of approximately 50 amino acid residues in length. These domains, designated as HD-I, HD-II and HD-III, are located between amino acid residues 160 and 204, 247 and 298, and 706 and 749, respectively. Further analysis has shown that the HD-I and HD-II domains can bind both to themselves and to one another. A domain was mapped in p127 between amino acid residues 377 and 438, that strongly reduces the degree of multimerization of chimaeric proteins containing HD-I and/or HD-II. Electron microscopy examination of negatively stained chimaeric proteins has shown that protein A fused with either the domain HD-II or the domain HD-III forms discrete structures consistent with the formation of quaternary complexes, whereas protein A fused to a non-self binding domain of p127 appears monomeric. These results indicate that p127 alone is able to build quaternary structures forming a network with which other proteins associate. As revealed by the tumorous phenotype resulting from the inactivation of the l(2)gl gene, the organization of the p127 network and its association with other proteins play critical roles in the control of cell proliferation (Jakobs, 1996).
Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribbled, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and the lack of phenotypes suggest that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types. In other words, Drosophila Amphiphysin functions in membrane morphogenesis, but an additional role in endocytosis cannot yet be dismissed (Zelhof, 2001).
To generate different cell types, some cells can segregate protein determinants into one of their two daughter cells during mitosis. In Drosophila neuroblasts, the Par protein complex localizes apically and directs localization of the cell fate determinants Prospero and Numb and the adaptor proteins Miranda and Pon to the basal cell cortex, to ensure their segregation into the basal daughter cell. The Par protein complex has a conserved function in establishing cell polarity but how it directs proteins to the opposite side is unknown. A principal function of this complex is to phosphorylate the cytoskeletal protein Lethal (2) giant larvae [Lgl; also known as L(2)gl]. Phosphorylation by Drosophila atypical protein kinase C (aPKC), a member of the Par protein complex, releases Lgl from its association with membranes and the actin cytoskeleton. Genetic and biochemical experiments show that Lgl phosphorylation prevents the localization of cell fate determinants to the apical cell cortex. Lgl promotes cortical localization of Miranda, and it is proposed that phosphorylation of Lgl by aPKC at the apical neuroblast cortex restricts Lgl activity and Miranda localization to the opposite, basal side of the cell (Betschinger, 2003).
How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. Polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell (Hutterer, 2004).
These results describe the first steps of a molecular pathway that leads to the establishment of polarity in epithelial cells of the Drosophila ectoderm. The Par-6 protein localizes to the apical cell cortex by binding to Cdc42. Par-6 recruits Bazooka and aPKC and is essential for establishment of the apical domain. Maintenance of Par-6 localization requires Lgl, a substrate of aPKC. Phosphorylation by aPKC inactivates Lgl at the apical cell cortex and restricts Lgl to the basolateral cortex to establish the basolateral domain (Hutterer, 2004).
Apical localization of Par-6 is a key event in the establishment of epithelial polarity. How is Par-6 recruited to the apical cell cortex? In C. elegans, the proteins Par-3, Par-6, and aPKC are localized to the anterior cell cortex before and during the first cell division. Their asymmetric localization is initiated by interaction of the sperm aster with the overlying cell cortex that excludes Par-6 from the posterior cell cortex. During Drosophila cellularization, centrosomes are located apically and it is therefore unlikely that a similar cortical microtubule interaction is responsible for the apical localization of Par-6 (Hutterer, 2004).
Although a distinct apical domain with sharp boundaries is established in epithelial cells only after cellularization, elegant membrane tracer experiments have revealed a subdivision of the plasma membrane into distinct regions already during cellularization. Are these membrane compartments prefiguring the future apical and basolateral domains and is Par-6 localizing apically by recognizing a preformed membrane domain? The first membrane domain is the furrow canal at the tip of the ingrowing cellularization front that is marked by Patj. This domain disintegrates after cellularization and is therefore unlikely to participate in Par-6 localization. During later stages, new membrane is preferentially inserted apically, then apicolaterally. At these stages, newly inserted membrane displaces the pre-existing membrane toward both the apical and basolateral side, indicating that a distinct apical membrane compartment is not established by the end of cellularization. It is therefore unlikely that Par-6 recognizes a preformed apical membrane compartment although these experiments do not rule out a more general role of the vesicle transport machinery in Par-6 localization (Hutterer, 2004).
The results indicate that Par-6 needs to bind to activated Cdc42 in order to localize apically. Since cdc42 mutants cannot be analyzed at this stage, a conserved proline in the CRIB domain was mutated to generate a Par-6 version that no longer binds Cdc42. The structure of the Par-6 Cdc42 complex shows that this residue comes to lie in a hydrophobic groove of the Cdc42 molecule. This may explain why it can be replaced by alanine without affecting Cdc42 binding. When it is deleted, however, one of the adjacent highly charged amino acids will occupy the position of the proline. This could strongly inhibit interaction with the hydrophobic pocket and eliminate binding to Cdc42 both in vertebrates and in flies. Since both Lgl and aPKC still bind Par-6-DeltaP and the protein is expressed at almost wild-type levels from the endogenous promoter in an otherwise null mutant background, par-6-DeltaP embryos are specifically defective in binding of Cdc42 to the Par-6/aPKC complex (Hutterer, 2004).
How does activated Cdc42 localize Par-6? Cdc42 might be required for association of an unidentified Par-6 binding partner that is essential for apical localization of the protein. The conformation of Par-6 changes upon binding to Cdc42, and this could affect interactions with other proteins. However, aPKC and Lgl are the only proteins identified in the Par-6 complex, and their interaction does not depend upon Cdc42 binding. In vertebrates, Par-6 interacts with the Stardust homolog Pals1, and this interaction is regulated by Cdc42. Stardust acts together with its binding partner Crumbs, but apical protein localization is initiated correctly in crumbs mutants. Therefore, it is unlikely that Stardust binding to Par-6 is critical for the initial apical localization of Par-6. It is more likely that Cdc42 activation provides an instructive cue for Par-6 localization. Cdc42 could be preferentially activated on the apical side, for example by localization of an exchange factor, and this could recruit Par-6 to the apical cell cortex. This hypothesis is supported by the ectopic patches of Par-6, which are observed after overexpression of constitutively active Cdc42. Asymmetric activation of Cdc42 is known to polarize other cell types. In yeast, the exchange factor Cdc24 is localized to the incipient bud site. This locally activates Cdc42 and polarizes the actin cytoskeleton toward the site. In migrating neutrophils, Cdc42 is locally activated in response to a chemoattractant gradient by the exchange factor PIXalpha. A clear Drosophila ortholog of PIXalpha exists, but whether it is involved in epithelial polarity remains to be determined (Hutterer, 2004).
Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Lgl acts at the basolateral cortex where it inhibits cortical localization of Par-6. How Lgl excludes Par-6 from the cortex is unclear, but it is remarkable that in other tissues, Lgl actually promotes cortical protein localization. In MDCK cells, Lgl was suggested to regulate basolateral exocytosis and it could recruit a Par-6 antagonizing factor to the basolateral plasma membrane. Since Lgl and Bazooka binding to Par-6 seem to be mutually exclusive, Lgl could also inactivate the Par protein complex by displacing Bazooka. To perform its role in epithelial polarity, Lgl needs to be phosphorylated by aPKC. This modification has been shown to inactivate the protein and release it from its association with membranes and the cytoskeleton. These results suggest that in epithelial cells, apically localized aPKC phosphorylates Lgl to displace the protein from the apical cell cortex. A simple model is proposed in which mutual inhibition between Par-6/aPKC on the apical and Lgl on the basolateral cell cortex maintains epithelial polarity. This model is in agreement with previous studies that demonstrate negative genetic interactions between lgl and proteins that localize to the apical domain. Furthermore, it provides a molecular explanation for the recently described suppression of the lgl mutant epithelial polarity phenotype by reduction of aPKC levels. Negative interactions between the apical and basolateral domains of epithelial cells have been described before. In the Drosophila follicular epithelium, Bazooka is phosphorylated and inhibited by Par-1, a protein kinase located on the basolateral domain, thus restricting the Par protein complex to the apical domain (Hutterer, 2004).
The proteins Par-6, Bazooka, and aPKC localize to the apical cell cortex of both neuroblasts and epithelial cells, but the mechanism of apical localization seems to be different in the two cell types. In epithelial cells, Lgl is required for maintaining Par proteins at the apical cell cortex, while Par protein localization in neuroblasts is Lgl independent. Expression of nonphosphorylatable Lgl disrupts asymmetric cell division in neuroblasts but is without effect in epithelial cells. In addition, overexpression of dominant-active or -negative Cdc42 disrupts epithelial polarity but has no effect on neuroblast division. What is the basis for these differences (Hutterer, 2004)?
Epithelial cells rely on adherens junctions for maintaining distinct membrane compartments. Such junctions are absent from neuroblasts, and in fact, distinct membrane compartments do not seem to exist. Instead, Par protein localization in neuroblasts requires a protein called Inscuteable that is recruited apically by binding to Bazooka and aPKC and activates heterotrimeric G proteins through an adaptor molecule called Pins. Both Inscuteable and G proteins are essential for maintaining Par protein localization in neuroblasts but not epithelial cells. It is possible that a feedback loop operates downstream of the G proteins to maintain polarity in the absence of diffusion barriers and cellular junctions. Mechanistic differences in the way Par proteins localize are also observed between species. In C. elegans, neither Lgl nor G proteins are required for Par-3 or Par-6 localization. Instead, a Ring finger protein called Par-2 maintains Par-3 and Par-6 at the anterior pole. Cdc42 plays a role, but only in maintenance and not establishment of polarity. Clearly, key players are missing that might help in an understanding of these mechanistic differences (Hutterer, 2004).
Cdc42 binds vertebrate Par-6. Both proteins are implicated in polarizing vertebrate epithelial cells, and their conserved interaction suggests that they achieve this via a conserved mechanism. Although in vertebrates both proteins primarily act on tight junctions, the role of Cdc42 in localizing the Par proteins seems conserved since overexpression of an activated form inhibits the localization of Par-3 to tight junctions in MDCK cells. However, current experiments do not confirm a previously demonstrated role of Cdc42 in activating Par-6-associated aPKC in vitro. Unlike in vertebrates, aPKC is shown to be equally active - at least toward Lgl - when bound to a form of Par-6 that does not interact with Cdc42. Whether species-specific differences or the different experimental setups are responsible for this apparent discrepancy remains unclear. Besides their function in polarity, the Par proteins are involved in proliferation control of vertebrate epithelial cells. Par-6 cooperates with Cdc42 in transforming cells, suggesting a role in oncogenic transformation. In Drosophila, Cdc42, Lgl, and Bazooka were shown to cooperate with activated ras in the formation of metastatic tumors. It can be anticipated that the powerful tool of Drosophila genetics will help to identify other components of this pathway that might clarify its role in carcinogenesis (Hutterer, 2004).
Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. A saturating genetic screen of the Drosophila autosomal genome was used to identify functional partners of dFmr1. Nineteen mutations were recovered in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. Direct evidence is provided for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. The data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex (Zarnescu, 2005).
This study reports the identification of Lgl as a functional partner of the Fragile X protein, Fmrp. Lgl forms a large macromolecular complex with Fmrp, which is developmentally regulated and modulates the architecture of the neuromuscular junction in the fly. At the cellular level, Lgl and Fmrp are temporally and spatially coexpressed during development and colocalize in granules in the soma and the developing neurites of mouse cultured cells. Fractionation experiments show that Fmrp and Lgl comigrate with Golgi membrane-associated complexes. Furthermore, the Fmrp/Lgl complex contains a subset of mRNAs and interacts physically and genetically with the PAR complex, an essential component of the cellular polarization pathway. These results suggest that Lgl functions with Fmrp to regulate a subset of target mRNAs during synaptic development and/or function. It is proposed that Lgl may regulate Fmrp/mRNA containing RNPs by (1) sorting at the Golgi, (2) transport in neurites, and (3) anchoring at specific membrane domains, such as subsynaptic sites. The neurite transport function may involve molecular motors such as myosin II and kinesin, previously shown to associate with Lgl and Fmrp, respectively. The anchoring mechanism may involve the PAR complex, which has a demonstrated role in defining membrane domains and has recently been shown to generate asymmetry in the C. elegans embryo by stabilizing RNPs at the posterior pole (Zarnescu, 2005).
The data demonstrate that Fmrp and Lgl form a functional complex in living neurons, and this is conserved in flies and mice. In the mouse brain, mFmrp associates with mLgl preferentially at a time of increased synaptogenesis, demonstrating a developmentally regulated interaction between Lgl and Fmrp. The data suggest that dLgl acts to regulate a subset of dFmr1-associated mRNAs with some encoding circadian regulated molecules (CG3348 and CG9681) and some encoding secreted or transmembrane proteins (CG6136, CG4101, CG9681) among others. dLgl also associates with mRNA independent of dFmr1; thus, it is formally possible that dLgl interacts with other RNA binding proteins, which remain to be determined (Zarnescu, 2005).
Fmrp has been implicated in the translational regulation of specific target mRNAs, perhaps via the RNAi pathway. Also, it has been proposed that Fmrp is involved in the transport and localization of mRNAs: cellular fractionation and immunolocalization data revealed the association of Fmrp-containing complexes with molecular motors such as kinesin and myosin V. Lgl functions in cellular polarity via regulating myosin motor activity and/or vesicle transport and has been shown to regulate polarized delivery by sorting at the Golgi. Taken together, these concepts suggest that Lgl may act as a scaffold for Fmrp granules, possibly at the Golgi, and perhaps aids in carrying specific mRNA targets to sites of locally controlled translation (such as subsynaptic sites) (Zarnescu, 2005).
It is also possible that Lgl anchors Fmrp at specific membrane domains such as synapses, perhaps via the PAR complex. Lgl function is regulated by the PAR complex, specifically via phosphorylation by aPKC-zeta and by direct binding to PAR6. The genetic interaction data suggest that PAR6 and Baz antagonize dFmr1 function, which is in accordance with previously published work showing that the PAR complex inhibits dlgl and with data that demonstrate that dlgl functions cooperatively with dFmr1. aPKC-zeta was shown to antagonize most dLgl functions with the exception of its role in regulating neuroblast apical size and the data are be consistent with such reports. Loss of function for aPKC-zeta suppresses gain of function sev:dFmr1 as well as the loss-of-function phenotype of dFmr1 at the NMJs. Taken together, these data suggest a dynamic relationship between the various members of the complex. One possible interpretation of the results is that aPKC/PAR can act on Fmrp directly or via Lgl depending on the developmental and/or cellular context. Furthermore, this is consistent with sucrose fractionation experiments, which suggest the existence of at least two complexes comprising dFmr1/PAR proteins and dFmr1/dLgl/PAR proteins (Zarnescu, 2005).
The PAR complex not only functions in cell polarity, but also at the synapse, where it is believed to function in synaptic tagging. Synaptic tags have been proposed to transiently mark a synapse after activation in a way that will translate the local events into persistent functional changes (such as long-term depression), processes in which Fmrp is also thought to act. Thus, the Fmrp/Lgl/PAR complex may act in synaptic plasticity linking synaptic input to the remodeling of the cytoskeleton and mediating required translational changes (Zarnescu, 2005).
Cell polarity and cell proliferation can be coupled in animal tissues, but how they are coupled is not understood. In Drosophila imaginal discs, loss of the neoplastic tumor suppressor gene scribble, which encodes a multidomain scaffolding protein, disrupts epithelial organization and also causes unchecked proliferation. Using an allelic series of mutations along with rescuing transgenes, domain requirements for polarity, proliferation control, and other Scrib functions have been identified. The leucine-rich repeats (LRR) tether Scrib to the plasma membrane, are both necessary and sufficient to organize a polarized epithelial monolayer, and are required for all proliferation control. The PDZ domains, which recruit the LRR to the junctional complex, are dispensable for overall epithelial organization. PDZ domain absence leads to mild polarity defects accompanied by moderate overproliferation, but the PDZ domains alone are insufficient to provide any Scrib function in mutant discs. A model is suggested in which Scrib, via the activity of the LRR, governs proliferation primarily by regulating apicobasal polarity (Zeitler, 2004).
These results highlight the central role of the LRR in Scrib function. Animals with absent or mutant LRR have phenotypes identical to those entirely lacking Scrib, with dramatic effects on both epithelial polarity and growth control. Of the five evolutionarily conserved protein-protein interaction domains in Scrib, only expression of the LRR can provide polarizing and proliferation-controlling activity, with high levels sufficient to effect nearly full rescue. The LRR is also sufficient to mediate membrane localization, whereas a protein lacking the LRR remains in the cytoplasm. Interestingly, alteration of a conserved leucine in the 10th LRR disrupts all Scrib functions and displaces the protein into the cytoplasm. A related alteration in the 13th LRR of C. elegans (Let-413) also prevents membrane localization and function. However, it is clear that the LRR functions as more than a membrane attachment domain because the Scrib PDZs alone are incapable of rescuing any aspect of the mutant phenotype, even when provided with an exogenous membrane targeting signal (Zeitler, 2004).
How can the LRR, which is broadly localized in the absence of PDZ domains, convey information to specifically polarize the apicobasal axis? It has been suggested that a critical role of Scrib in epithelial polarity is the recruitment of Lgl to the lateral cell cortex. Lgl itself is not highly polarized in its distribution, and while it is displaced from the cortex in scrib null GLC embryos, immunofluorescense reveals that Lgl is indeed cortically localized in LRR-expressing scrib 4 GLC embryos, consistent with proper apicobasal polarization in these animals. Therefore, it appears that membrane-localized LRR can mediate interactions that effect cortical recruitment of Lgl, where Lgl can perform its still unknown activities in regulating protein trafficking (Zeitler, 2004).
In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Partner of numb (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. This study used 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. It was demonstrated 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. It is proposed 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 (Mayer, 2005).
In order to study the dynamics of asymmetric protein localization, a time series of the division of an SOP cell expressing GFP-Pon and Histone2B-RFP was recorded under the control of a specific promoter. Histone2B-RFP was used to visualize DNA, thus allowing correlation of distinct steps of GFP-Pon localization with other mitotic events. In interphase, some GFP-Pon is cortical, but a large part localizes to the cytoplasm. As the cell enters mitosis, it rounds up and undergoes strong membrane blebbings, indicative of local rearrangements of the cortical cytoskeleton. Interestingly, similar blebbing events have also been observed in the first division of the C. elegans zygote. Unlike in SOP cells, however, they only occur on the anterior side of the C. elegans zygote, where Par-3/6 localize. Shortly after blebbing has started, chromosomes condense and GFP-Pon accumulates on random sites of the cell cortex. The accumulations are transient and do not necessarily predict the position of the final Pon crescent. This suggests that the process leading to Pon accumulation can take place all around the cell but is reinforced specifically in the crescent region. Some GFP-Pon was also observed at the nucleus. This signal might be due to GFP-Pon binding to the nuclear envelope or to the endoplasmic reticulum, and it disappears slowly after nuclear-envelope breakdown. At nuclear-envelope breakdown, cortical blebbing ceases, the cell cortex smoothes, and first signs of asymmetric localization of GFP-Pon into an anterior cortical crescent are observed. As the cell progressed into metaphase, the GFP-Pon signal in the crescent area becomes stronger. Surprisingly, the intensity of the cortical area opposite of the crescent is almost not changed during this process. Thus, GFP-Pon might actually be recruited to the crescent directly from the cytoplasm rather than being transported along the cell cortex. Indeed, quantification of fluorescence intensity showed that GFP-Pon recruitment at the cell cortex is accompanied by a comparable loss of cytoplasmic GFP-Pon. Note that local degradation of GFP-Pon in the cytoplasm is not responsible for this reduction because total GFP-Pon remains unchanged (Mayer, 2005).
Subsequently, the metaphase plate was oriented with respect to the crescent, and during cytokinesis, GFP-Pon segregated largely into the anterior daughter cell. It is proposed that GFP-Pon localization is a two-step process involving the establishment of a cortical area where the crescent will form and the progressive recruitment of protein to the predefined site until metaphase (Mayer, 2005).
Asymmetry of Numb and Pon could be created by lateral movement along the cell cortex or by direct recruitment from the cytoplasm to one side of the cell cortex. To quantify the exchange of Numb and Pon between the cell cortex and the cytoplasm, fluorescence recovery after photobleaching (FRAP) was used of GFP fusions to Numb and Pon. Numb-GFP can partially rescue the numb mutant phenotype, indicating that it is functional. GFP-Pon contains just the asymmetric-localization domain. Its rescue behavior is unknown, but it colocalizes with endogenous Pon throughout mitosis. When cytoplasmic GFP-Pon is photobleached, fluorescence recovers with a half-time of 0.48 s, indicating that diffusion is not limiting. Recovery of cortical GFP-Pon fluorescence occurred with single exponential kinetics and a half-time of 35 s, whereas the half-time for Numb-GFP was 27 s. Therefore, Numb and Pon showed a surprisingly dynamic association with the cell cortex (Mayer, 2005).
Either cortical recruitment of cytoplasmic GFP-Pon or lateral diffusion/transport of cortical GFP-Pon could be responsible for fluorescence recovery. To measure the exchange between cortical and cytoplasmic Pon, an area covering approximately 40% of the cytoplasm was repeatedly photobleached in an SOP cell expressing GFP-Pon. Fluorescence intensity was simultaneously recorded at the cortex. Cortical fluorescence intensity dropped to less than 5% with a half-time of 52 s. Thus, the cortical and cytoplasmic pools of GFP-Pon rapidly interchange with a mobile fraction of more than 95% (Mayer, 2005).
When the dynamic association with the cell cortex is taken into account, Pon asymmetry could be explained either by fast and continuous lateral transport or by directed recruitment to an asymmetric cortical binding site. To determine the contribution of lateral transport, FRAP rates were compared on the edge and in the center of a photobleached region within the GFP-Pon crescent. The bleached region was defined such that a region of nonbleached molecules was left behind at the edges of the crescent after photobleaching. To avoid recovery from above and below the image plane, a protocol was used in which the region of interest was bleached in several planes. The efficiency of this procedure was confirmed by 3D reconstruction after photobleaching in fixed tissue. FRAP curves from ten experiments were averaged. Their superposition shows that the two regions recover nearly identically with half-times of 32 s for a region close to nonbleached GFP-Pon and of 35 s for a region farther away. Taken together, these observations suggest a model where Pon is preferentially recruited from the cytoplasm to the site of crescent formation. It is proposed that a cortical high-affinity binding site for Pon is established during mitosis and mediates specific recruitment of Pon to one side of the cell cortex (Mayer, 2005).
To test the role of Lgl in asymmetric protein localization in SOP cells, cortical recruitment of GFP-Pon was measured in lgl1 mutant clones. In a similar experiment, Lgl has been shown to be dispensable for Pon localization, although Pon recruitment seemed to be delayed. The ratio between total cortical and total cytoplasmic fluorescence was calculated. Because GFP fluorescence intensity is proportional to GFP-Pon concentration, this ratio should give a good estimate of the fraction of GFP-Pon localized at the cell cortex. Although GFP-Pon was still asymmetric, quantitative analysis revealed that the cortical GFP-Pon fraction was slightly but significantly reduced in lgl1 mutant clones. This might be a hypomorphic phenotype caused by small residual amounts of Lgl protein present in the mutant clones. Therefore expression of deregulated aPKC (aPKC-deltaN) was used as another means to inactivate Lgl. Expression of aPKC-deltaN was shown to phenocopy lgl mutants in embryonic tissues, presumably because it phosphorylates and inactivates Lgl all around the cell. In contrast to lgl1 mutant SOP cells, a much stronger decrease of cortical GFP-Pon recruitment was observed upon aPKC-deltaN expression. Still, a slight cortical asymmetry was observed, which is thought is due to the presence of endogenous aPKC. Even at anaphase, the degree of recruitment hardly reached that of control cells in prophase. To test whether Lgl phosphorylation was responsible for this phenotype, aPKC-deltaN was coexpressed with nonphosphorylatable lgl3A. Expression of lgl3A completely rescued the cortical-recruitment defect. The observed differences are not due to increased protein levels because total cellular GFP-Pon fluorescence remains constant (Mayer, 2005).
Thus, active, nonphosphorylated Lgl is needed for cortical recruitment of GFP-Pon although lgl1 mutant clones did not show a very strong phenotype. The easiest explanation for the discrepancy between the lgl1 mutant and ectopic Lgl phosphorylation is the perdurance of residual Lgl protein in mutant tissue. This is supported by previous observations describing Numb-localization defects in temperature-sensitive alleles of lgl. It is possible that Lgl can mediate its effects even at protein concentrations below the detection limit of the antibody. Thus, Lgl may not be needed at stoichiometric levels for asymmetric protein localization in SOP cells, but it instead plays a catalytic or signaling role (Mayer, 2005).
How could Lgl recruit Pon to the cell cortex? Formally, it is possible that Pon simply binds Lgl in a phosphorylation-dependent manner. However, no direct interaction has been described and such a model would not explain why Pon is cortical even when Lgl levels are strongly reduced. Two other models are more likely: Either cortical binding sites for Numb and Pon are present all around the cell, but their affinity depends on Lgl and its phosphorylation status and therefore varies along the cell cortex (Model 1); or a limiting number of cortical binding sites are present only on one side of the cell, and Lgl is responsible for their asymmetric distribution (Model 2). To distinguish between these models, FRAP rates were measured for cortical GFP-Pon in different genetic backgrounds. The FRAP rate is a function of the rate constants for both association and dissociation of GFP-Pon with its postulated cortical binding site. In Model 1, expression of activated lgl (lgl3A) or deregulated aPKC (aPKC-?N) should alter the affinity of the binding site and therefore change the rate constants, resulting in a variation of the FRAP rate. Because the FRAP rate is independent of receptor concentration, however, it would remain constant under the same conditions in Model 2. Cortical GFP-Pon FRAP rates were measured in wild-type SOP cells, in cells expressing lgl3A, and in cells where Lgl was inactivated by expression of aPKC-deltaN. Although expression of aPKC-deltaN dramatically reduced the amount of GFP-Pon present at the cortex, it did not influence the kinetics of GFP-Pon binding to the cortical binding site. Thus, the number of Pon binding sites at the cell cortex, and not their affinity for Pon, seems to be reduced by aPKC-deltaN expression (Mayer, 2005).
To gain independent evidence for the two models, the fraction of GFP-Pon present at the cell cortex was quantitated. If Lgl regulated GFP-Pon binding site affinity, expression of lgl3A would change the entire SOP cell cortex to high affinity, and therefore it would increase the cortical GFP-Pon fraction. If Lgl regulated only the distribution of binding sites, however, the cortical fraction of GFP-Pon should remain the same. Cortical recruitment was quantified by measuring the ratio of cortical to cytoplasmic fluorescence for GFP-Pon and Numb-GFP at different time points in mitosis. Compared to wild-type cells, expression of lgl3A did not cause a significant increase in cortical recruitment. This is not because cytoplasmic GFP-Pon is limiting; increased GFP-Pon expression predominantly increased the cytoplasmic signal. Taken together, these results favor Model 2, in which Lgl acts by asymmetrically distributing a limiting number of cortical GFP-Pon binding sites. The loss of cortical fluorescence upon aPKC-deltaN expression indicates that lgl is also required for binding site formation, in addition to binding site positioning. However, this second role of lgl does not seem to be rate limiting under normal conditions because lgl3A expression does not increase the cortical GFP-Pon fraction. Although these results are most consistent with Model 2, more-complex models cannot be excluded. For example, lgl could distribute a limiting adaptor protein that links Pon to a receptor but is not the receptor itself (Mayer, 2005).
The direct cortical binding partners for Pon or Numb have not yet been identified. Thus, it is only possible to speculate on the molecular mechanisms of their postulated asymmetric distribution. Although the results are inconsistent with lateral transport of GFP-Pon, they do not exclude lateral transport of its cortical anchor. Similar to what has been proposed for asymmetric cell division in C. elegans, a possible mechanism could be local tearing and contraction of the cortical actin cytoskeleton. Lgl was shown to inhibit the cortical localization of myosin II, and it has been proposed that cortical myosin II might exclude asymmetrically segregating proteins. These data could be integrated with the model if myosin II excludes the cortical binding sites rather than influencing determinant localization directly. Alternatively, transmembrane receptors for Pon or Numb could be delivered to the position of crescent formation by vesicle transport. Such a mechanism in which transmembrane receptors are present on vesicles that dock at the membrane in an Lgl-dependent fashion would be consistent with the quantitative observations. It would also explain why Lgl is essential for crescent formation but not needed in metaphase for maintenance of asymmetric protein localization. It is remarkable that lateral diffusion of transmembrane proteins is slow enough to allow a stable asymmetric distribution, if the delivery of the protein is asymmetric, both in yeast and in SOP cells. The yeast Lgl orthologs Sro7p and Sro77p have been implicated in plasma-membrane fusion of secretory vesicles (Lehman, 1999), and Lgl has been proposed to regulate vesicular targeting to specific membrane domains. Furthermore, asymmetric protein localization in Drosophila requires myosin VI, a motor whose main function is vesicle movement, suggesting that vesicle trafficking plays some role (Mayer, 2005).
These data provide insight into the dynamic protein movements of cell-fate determinants and their associated adaptor proteins during asymmetric cell division. It is proposed that these determinants are preferentially recruited from the cytoplasm to a high-affinity binding site during late prophase. Establishment of this binding site is regulated by the phosphorylation status of Lgl. The role of Lgl is more to concentrate binding sites on one side of the cell than to act as a receptor itself or change the affinity of another Numb or Pon binding site (Mayer, 2005).
Drosophila neural precursor cells divide asymmetrically by segregating the Numb protein into one of the two daughter cells. Numb is uniformly cortical in interphase but assumes a polarized localization in mitosis. This study shows that a phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis. Aurora-A phosphorylates Par-6, a regulatory subunit of atypical protein kinase C (aPKC). This activates aPKC, which initially phosphorylates Lethal (2) giant larvae (Lgl), a cytoskeletal protein that binds and inhibits aPKC during interphase. Phosphorylated Lgl is released from aPKC and thereby allows the PDZ domain protein Bazooka to enter the complex. This changes substrate specificity and allows aPKC to phosphorylate Numb and release the protein from one side of the cell cortex. These data reveal a molecular mechanism for the asymmetric localization of Numb and show how cell polarity can be coupled to cell-cycle progression (Wirtz-Peitz, 2008).
Since the discovery of Numb asymmetry, several proteins required for Numb localization have been identified, but how they cooperate remained unclear. This paper describes a cascade of interactions among these proteins that culminates in the asymmetric localization of Numb in mitosis. In interphase, Lgl localizes to the cell cortex, where it forms a complex with Par-6 and aPKC. At the onset of mitosis, AurA phosphorylates Par-6 in this complex, thereby releasing aPKC from inhibition by Par-6. Activated aPKC phosphorylates Lgl, causing its release from the cell cortex. Since Baz competes with Lgl for entry into the Par complex, the disassembly of the Lgl/Par-6/aPKC complex allows for the assembly of the Baz/Par-6/aPKC complex. Baz is a specificity factor that allows aPKC to phosphorylate Numb on one side of the cell cortex. Since p-Numb is released from the cortex (Nishimura, 2007; Smith, 2007), these events restrict Numb into a cortical crescent on the opposite side (Wirtz-Peitz, 2008).
The data show that Lgl acts as an inhibitory subunit of the Par complex. Given that Par-6 inhibits aPKC activity until the onset of mitosis, why would an additional layer of regulation be required? Like all phosphoproteins Numb is in a dynamic equilibrium between the phosphorylated and unphosphorylated states. Too high a rate of phosphorylation shifts this equilibrium toward the phosphorylated state, mislocalizing Numb into the cytoplasm. Too low a rate shifts it toward the unphosphorylated state, mislocalizing Numb around the cell cortex. Importantly, these data show that only the Baz complex can phosphorylate Numb. Assuming an abundance of Lgl over cortical Par-6, an increase in aPKC activity would translate into a comparatively small increase in the levels of Baz complex. This is because assembly of the Baz complex requires free subunits of Par-6 and aPKC, which become available only once the pool of cortical Lgl has been completely phosphorylated. Therefore, it is proposed that Lgl acts as a molecular buffer for the activity of the Par complex toward Numb. This maintains Numb phosphorylation within a range that is sufficiently high to exclude Numb from one side of the cell cortex but sufficiently low to permit the cortical localization of Numb to the other side (Wirtz-Peitz, 2008).
What is the evidence for this model? Lgl3A, a nonphosphorylatable mutant of Lgl in which the three aPKC phosphorylation sites are mutated to Ala, infinite buffering capacity, induces the mislocalization of Numb around the cell cortex. Conversely, in lgl mutants, having no buffering capacity, Numb is mislocalized into the cytoplasm. Moreover, the model predicts the loss of buffering capacity in the lgl mutant to be offset by an increase in the amount of substrate, since this would render the excess activity of the Par complex limiting. Indeed, overexpression of Numb in lgl mutants restores the cortical localization of Numb as well as its cortical asymmetry (Wirtz-Peitz, 2008).
The results indicate that Lgl gain- and loss-of-function phenotypes are entirely accounted for by the role of Lgl in inhibiting the assembly of the Baz complex. Previously, however, it was thought that the asymmetric phosphorylation of Lgl by aPKC restricts an activity of Lgl to the opposite side of the cell cortex. Based on this model, it was subsequently proposed that Lgl mediates the asymmetric localization of cell fate determinants by inhibiting the cortical localization of myosin-II. In addition, the role of the yeast orthologs of Lgl in exocytosis led to speculation that Lgl establishes an asymmetric binding site for cell fate determinants by promoting targeted vesicle fusion. However, the data show that Lgl asymmetry is extremely transient, and that the protein is completely cytoplasmic from NEBD onward. Lgl cannot therefore interact with any cortical proteins in prometaphase or metaphase, when myosin-II was reported to localize asymmetrically, or establish a stable landmark for vesicle fusion. Interestingly, a recent study demonstrated that yeast Lgl inhibits the assembly of SNARE complexes by sequestering a plasma membrane SNARE (Hattendorf, 2007). This mechanism is reminiscent of fly Lgl sequestering Par-6 and aPKC from interaction with Baz, suggesting that the defining property of Lgl-family members is not a specific role in exocytosis, but a more generic role in regulating the assembly of protein complexes (Wirtz-Peitz, 2008).
The data identify Numb as a key target of aPKC in tumor formation and suggest that Lgl acts as a tumor suppressor in the larval brain by inhibiting the aPKC-dependent phosphorylation of Numb. Although it is tempting to conclude that tumor formation in lgl mutants results from the missegregation of Numb, missegregation of Numb in numbS52F or upon expression of Lgl3A does not cause neuroblast tumors. How might this be explained? During mitosis, unphosphorylated cortical Numb is inherited by the differentiating daughter. At the same time, Baz and aPKC are excluded from this daughter, which limits Numb phosphorylation after exit from mitosis. In the subsequent interphase, some differentiating daughters reexpress members of the Baz complex (Bowman, 2008), but Numb continues to be protected from phosphorylation since cortical Lgl prevents the reassembly of the Baz complex. Thus, Lgl acts both in mitosis and interphase to maximize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).
In lgl mutants, Numb phosphorylation is increased in mitosis, and less unphosphorylated Numb is segregated into the basal daughter cell. Moreover, the assembly of the Baz complex is unrestrained in the subsequent interphase, which is exacerbated by the missegregation of aPKC into both daughter cells. Together, these defects minimize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).
Why is the amount of unphosphorylated Numb critical for differentiation? Recently, it was shown that aPKC-dependent phosphorylation of Numb inhibits not only its cortical localization, but also its activity, owing to the reduced affinity of p-Numb for its endocytic targets (Nishimura, 2007). Therefore, ectopic phosphorylation of Numb leads to its inactivation, transforming the basal daughter cell into a neuroblast in a manner similar to mutation of numb. Consistent with this model, studies in SOP cells have documented ectopic Notch signaling in lgl mutants. Although the numbS52F mutant and Lgl3A overexpression also lead to missegregation of Numb, the levels of active unphosphorylated Numb are increased rather than decreased in these cases and are sufficient to support differentiation (Wirtz-Peitz, 2008).
The data also provide additional insight into the mechanism of tumor formation in aurA mutants. In aurA mutants, the differentiating daughter cell inherits less Numb because Numb is mislocalized around the cell cortex. At the same time, aPKC is missegregated into the differentiating daughter cell, where it promotes Numb phosphorylation in the subsequent interphase. Together, these events result in subthreshold amounts of unphosphorylated Numb in some basal daughter cells, transforming these into neuroblasts. This model explains why aurA mutants are characterized by reduced aPKC activity in mitosis, but are nonetheless suppressed by aPKC mutations, since a lack of aPKC in the differentiating daughter cell restores threshold amounts of unphosphorylated Numb (Wirtz-Peitz, 2008).
The data reveal that Lgl inhibits Numb phosphorylation to maintain Numb activity, whereas AurA promotes Numb phosphorylation in mitosis to ensure its asymmetric segregation. It is concluded that Lgl and AurA act on opposite ends of a regulatory network that maintains appropriate levels of Numb phosphorylation at the appropriate time in the cell cycle (Wirtz-Peitz, 2008).
Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC -| Lgl -| myosin II -| Miranda). This study provides biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement, suggesting that the repressive-cascade model is incorrect. In investigating key results that led to this model, it was found that Y-27632, a Rho kinase inhibitor used to implicate myosin II, efficiently inhibits aPKC. Lgl3A, a nonphosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. It is concluded that Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, Lgl instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown (Atwood, 2009).
This study examined the mechanism by which polarity is generated in Drosophila neuroblasts, a process required for the segregation of cell fate determinants during asymmetric cell division. This process utilizes aPKC, which is found in many polarized systems such as epithelia. Previously, polarization of the protein Miranda, which is normally restricted to the basal neuroblast cortex opposite aPKC, has been thought to occur by a complex cascade of repressive interactions involving the tumor suppressor Lgl and the motor protein myosin II. The finding that aPKC phosphorylation displaces Miranda from the cortex of neuroblasts and S2 cells led to the idea that the repressive-cascade model might not accurately describe Miranda displacement. This prompted a reexamination of key results supporting the repressive-cascade model (Atwood, 2009).
Based on previous results, it is proposed that studies suggesting that myosin II is involved in aPKC-mediated cortical displacement of Miranda are an artifact of inhibition of aPKC by the Rho kinase inhibitor Y-27632. Although the possibility cannot be excluded that the Miranda polarity defects observed in Y-27632-treated embryos are indeed the result of myosin II inhibition, the fact that this phenotype is identical to that exhibited by apkc mutants, the efficient inhibition of aPKC, and the high concentrations of this compound used in previous reports (~50 mM, compared to the IC50 < 10 μM for aPKC and 0.1 μM for Rho kinase) indicate that the simplest interpretation of the Y-27632 phenotype is direct inhibition of aPKC activity. The role of myosin II in Miranda cortical displacement, if any, is unclear (Atwood, 2009).
The central result that led to the placement of Lgl between aPKC and Miranda was reexamined. Expression of a form of Lgl in which the aPKC phosphorylation sites have been inactivated results in uniformly cortical Miranda in neuroblasts. This result can be interpreted in one of two ways: Lgl mediates Miranda cortical targeting and phosphorylation of Lgl represses this activity, or Lgl inhibits aPKC and this inhibition is repressed by aPKC phosphorylation (i.e., feedback). The key distinction between these two models is whether or not aPKC is repressed when Lgl3A is expressed. Several recent studies indicate that Lgl is a potent inhibitor of aPKC activity. Consistent with this, it was found that Lgl3A expression dramatically reduces the localization of aPKC to the neuroblast apical cortex. Furthermore, it was found that a form of aPKC that is not efficiently repressed by Lgl can overcome the effects of Lgl3A and drive Miranda into the cytoplasm, consistent only with Lgl phosphorylation not being a requirement for Miranda cortical displacement. In addition, it was shown that Lgl alone is sufficient for inhibition of aPKC activity. Thus, it is concluded that Lgl can directly inhibit aPKC and is not required for Miranda cortical targeting (Atwood, 2009).
A simpler mechanism than the repressive-cascade model for Miranda polarization by aPKC is favored: aPKC phosphorylates Miranda, causing it to be displaced from the cortex. The identification of Miranda as a direct aPKC substrate, the requirement of these phosphorylation events for cortical displacement in both S2 cells and neuroblasts, and the necessity of these phosphorylation events for normal development and viability support this model. The sufficiency of phosphorylation (phosphomimetic Miranda is cytoplasmic in the absence of aPKC) indicates that other phosphorylation events (such as phosphorylation of Lgl in the repressive-cascade model) are not required for Miranda cortical displacement. This new model dramatically simplifies the understanding of how asymmetric aPKC activity, as generated by Baz and Cdc42, is translated into the segregation of cell fate determinants. Thus, polarization of three components downstream of aPKC, Numb, and Miranda (this work), appears to occur by direct aPKC phosphorylation. Further work will be required to determine whether this mechanism is utilized by all factors that are polarized by aPKC (Atwood, 2009).
When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. This study shows that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. It was also shown that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. These observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration (Sun, 2011).
Many tissues have the capacity respond to the removal or death of cells by increasing proliferation of the remaining cells. In Drosophila, this phenomenon has been characterized both in the context of imaginal disc regeneration and compensatory cell proliferation. These studies implicate the Hippo signaling pathway as a key player in these proliferative responses to tissue damage. After genetically ablating the wing primordia by inducing apoptosis, it was observed that Yki becomes activated to high levels in surrounding cells, based on its nuclear abundance and induction of a downstream target of Yki transcriptional activity. Moreover, high level Yki activation is crucial for wing disc regeneration, as even modest reduction of Yki levels, to a degree that has only minor effects on normal wing development, severely impaired wing disc regeneration. While it was known that Yki is required for wing growth during development, the current observations establish that Yki is also required for wing growth during regeneration, and moreover that regeneration requires higher levels of Yki activation than during normal development (Sun, 2011).
These studies identify Jnk activation as a promoter of Yki activity in the wing disc. Most aspects of imaginal disc development, including imaginal disc growth, normally do not require Jnk signaling. By contrast, Jnk signaling is both necessary and sufficient for Yki activation in response to wing damage. Jnk signaling has previously been linked to compensatory cell proliferation and regeneration in imaginal discs, and it is now possible to ascribe at least part of that requirement to activation of Yki. However, Jnk signaling also promotes the expression of other mitogens, including Wg, which were linked to regeneration and proliferative responses to apoptosis. Wg and Yki are not required for each other's expression, suggesting that they are regulated and act in parallel to influence cell proliferation after tissue damage. The mechanism by which Jnk activation induces Yki activation is not yet known. The observation that it could be suppressed by over-expression of Wts or Hpo suggests that it might impinge on Hippo signaling at or upstream of Hpo and Wts, but the possibility that Jnk-dependent Yki regulation occurs in parallel to these Hippo pathway components cannot be excluded. The high level of nuclear Yki localization is striking by contrast with the more modest effects of upstream tumor suppressors in the Hippo pathway, which suggests that Jnk might regulate Yki through a distinct mechanism, or simultaneously affect multiple upstream regulators (Sun, 2011).
Strong Yki activation was detected within the wing and haltere discs in response to Jnk activation, but weaker or non-existent effects in leg or eye discs. Jnk activation has previously been linked to oncogenic effects of neoplastic tumor suppressors in eye discs, and it is possible that Yki activation might be induced in eye discs if a distinct Jnk activation regime were employed. Nonetheless, since identical conditions were employed in both wing and eye discs, isolating them from the same animals, these studies emphasize the importance of context-dependence for Yki activation by Jnk. A link between Jnk activation and Yki activation is not limited to the wing however, as a connection between these pathways was recently discovered in the adult intestine, where damage to intestinal epithelial cells, and activation of Jnk, can activate also Yki (Sun, 2011).
There was a general correspondence between activation of Jnk and activation of Yki under multiple experimental conditions, including expression of Rpr, direct activation of Jnk signaling by Egr or Hep.CA (an activated form of the Jnk kinase Hemipterous), and depletion of lgl. Some experiments, most notably direct activation of Jnk by Hep.CA, revealed a non-autonomous effect on Yki, which could imply that the influence of Jnk on Yki activity is indirect. Although the basis for this non-autonomous effect is not yet known, the hypothesis that it is actually also mediated through Jnk signaling is favored, since it has been reported that Jnk activation can propagate from cell to cell in the wing disc. Consistent with this possibility, a non-autonomous activation of Jnk adjacent to lgl depleted cells was seen to be blocked by depletion of bsk solely within the lgl RNAi cells. Conversely, alternative signals previously implicated in compensatory cell proliferation do not appear to be good candidates for mediating Yki activation, since it was found that Wg is not required for Yki activation in regenerating discs, and prior studies did not detect a direct influence of Dpp pathway activity on Yki activation (Sun, 2011).
Activation of Yki adjacent to Egr- or Rpr-expressing cells was also reduced by over-expression of Wts. This might reflect an influence of Yki on signaling from these cells, but because expression of Wts inhibits Yki activity, and activated Yki promotes expression of an inhibitor of apoptosis (Diap1), it is also possible that this effect could be explained simply by Wts over-expression resulting in reduction or more rapid elimination of Egr- or Rpr-expressing cells; the reduced survival of these cells would then limit their ability to signal to neighbors (Sun, 2011).
Although Jnk has been implicated in compensatory cell proliferation and regeneration, it is better known for its ability to promote apoptosis. The dual, opposing roles of Jnk signaling as a promoter of apoptosis and a promoter of cell proliferation raise the question of how one of these distinct downstream outcomes becomes favored in cells with Jnk activation (see Diverse inputs and outputs of Jnk signaling). Given the links between Jnk activation and human diseases, including cancer, defining mechanisms that influence this is an important question, and the identification of the role of Yki activation in Jnk-mediated proliferation and wing regeneration should facilitate future investigations into how the balance between proliferation or apoptosis downstream of Jnk is regulated (Sun, 2011).
Hippo signaling is regulated by proteins that exhibit discrete localization at the subapical membrane, e.g., Fat, Ex, and Merlin. The observation that disruption of apical-basal polarity is associated with disruption of Hippo signaling underscores the importance of this localization to normal pathway regulation. These observations establish that Hippo signaling is inhibited by neoplastic tumor suppressor mutations, resulting in Yki activation, and that this activation of Yki is required for the tumorous overgrowths associated with these mutations (Sun, 2011).
Although these results agree with these recent studies in linking lgl to Hippo signaling (Grzeschik, 2010), there are some notable differences. A previous study examined lgl mutant clones in the eye imaginal disc, under conditions where cells retained apical-basal polarity, whereas this study examined wing imaginal discs, where apical-basal polarity was lost. Intriguingly this study found that conditions associated with activation of Yki by Jnk in the wing disc were not sufficient to activate Yki in the eye disc. This observation, together with the discovery that loss of polarity in lgl depleted wing cells requires Jnk activation, suggests as a possible explanation for why lgl null mutant clones retain apical-basal polarity in eye discs, that eye disc cells have a distinct, and apparently reduced, sensitivity to Jnk activation as compared to wing disc cells (Sun, 2011).
This study also identified distinct processes linked to Yki activation in the absence of lgl. A previous study reported an effect of lgl on Hpo protein localization (Grzeschik, 2010). In wing discs, the discrete apical localization of Hpo was observed in studies of eye discs. Thus, the proposed mechanism, involving activation of Yki via mis-localization of Hpo and dRassf, might not be relevant to the wing. By contrast, this study identified an essential role for Jnk signaling in regulating Yki activation in lgl-depleted cells in the wing. Because this study did not detect an effect of direct Jnk activation on Yki in eye discs, it is possible that Lgl can act through multiple pathways to influence Yki, including a Jnk-dependent pathway that is crucial in the wing disc, and a Jnk-independent pathway that is crucial in the eye disc. Grzeschik (2010) also linked the influence of lgl in the eye disc to its antagonistic relationship with aPKC. The observation that the influence of aPKC in the wing depends on Jnk activation is consistent with an Lgl-aPKC link, and identifies a role for Jnk activation in the oncogenic effects of aPKC (Sun, 2011).
The observation that the loss of polarity in lgl RNAi discs is dependent upon Jnk signaling was unexpected, but a related observation was recently reported by Zhu; 2010). These results suggest that the established role of the Lgl-Dlg-Scrib complex in maintaining epithelial polarity depends in part on repressing Jnk activity. However, since Jnk activation on its own was not sufficient to disrupt polarity, multiple polarity complexes might need to be disturbed in order for wing cells to lose apical-basal polarity, including both Lgl and additional, Jnk-regulated polarity complexes (Sun, 2011).
The discovery of the role of Jnk signaling in Yki activation provides a common molecular mechanism for the overgrowths observed in conjunction with mutations of neoplastic tumor suppressors, and those associated with compensatory cell proliferation, because in both cases a proliferative response is mediated through Jnk-dependent activation of Yki. Although the molecular basis for the linkage of these two pathways is not understood yet, it operates in multiple Drosophila organs, and thus appears to establish a novel regulatory input into Hippo signaling that is of particular importance in abnormal or damaged tissues. Moreover, Jnk activation has also been observed in conjunction with regeneration of disc fragments after surgical wounding, and thus its participation in regeneration is not limited to paradigms involving induction of apoptosis. It is also noteworthy that under conditions of widespread lgl depletion (i.e., lgl mutant or lgl RNAi), and consequent Jnk activation, the balance between induction of apoptosis and induction of cell proliferation is shifted towards a proliferative response. By contrast, in the wing disc clones of cells mutant for lgl fail to survive, unless oncogenic co-factors are co-expressed. The loss of lgl mutant clones in wing discs was recently attributed to cell competition. Together, these observations suggest that the choice between proliferative versus apoptotic responses to Jnk activation can be influenced by the Jnk activation status of neighboring cells (Sun, 2011).
The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl (Morais-de-Sa, 2014).
Very little is known about how the posterior follicle cells signal to polarise the AP axis of the oocyte, except that signalling is disrupted when the germline is mutant for components of the exon junction complex, such as Mago nashi. The current results reveal that Slmb also plays an essential role in this pathway, where it acts to establish the complementary cortical domains of Baz/Par-6/aPKC and Par-1. Although Slmb might act in a variety of ways to establish this asymmetry, the observation that it regulates the levels of the Par-6/aPKC complex suggests a simple model in which Slmb directly or indirectly targets a component of the complex for degradation at the posterior of the oocyte. Since aPKC phosphorylates Par-1 to exclude the latter from the cortex, the degradation of aPKC would allow the posterior recruitment of Par-1, which would then maintain polarity by phosphorylating and antagonising Baz. Indeed, this might explain the observation that Par-6 is excluded from the posterior cortex before Baz. The polarisation of the oocyte therefore appears to occur in two phases. During the initiation phase, Slmb removes the Par-6/aPKC complex from the posterior cortex to allow the recruitment of Par-1. Par-1 then maintains and reinforces this asymmetry by phosphorylating Baz to exclude it from the posterior cortex, thereby removing the cortical anchor for the Par-6/aPKC complex (Morais-de-Sa, 2014).
Slmb is usually recruited to its targets by binding to phosphorylated residues that lie 9-14 amino acids downstream from the ubiquitylated lysine. Although both aPKC and Par-6 contain several sequences that could serve as atypical Slmb binding sites, neither contains a classic Slmb-dependent degron sequence. It is therefore unclear whether the SCFSlmb complex directly ubiquitylates either protein to target it for degradation or whether it targets another, unknown component of the complex that is required for the stability of Par-6 and aPKC. Nevertheless, this model leads to the prediction that the polarising signal from the follicle cells will induce the activation of a kinase that phosphorylates a Slmb substrate at the posterior of the oocyte, thereby triggering the local degradation of the Par-6/aPKC complex (Morais-de-Sa, 2014).
The demonstration that Slmb is required for the exclusion of the Par-6/aPKC complex from the posterior of the Drosophila oocyte raises interesting parallels with AP axis formation in C. elegans. Although Drosophila does not have an equivalent of the main symmetry-breaking step in the worm, in which a contraction of the actomyosin cortex removes the anterior PAR proteins from the posterior, the function of Slmb is analogous to that of PAR-2 in the alternative polarity induction pathway. Both proteins act to remove the Par-6/aPKC complex from the posterior cortex to allow the posterior recruitment of Par-1, which then reinforces polarity by excluding Baz/PAR-3 by phosphorylation. Furthermore, the polarity phenotypes of both slmb and par-2 mutants can be rescued by the overexpression of Lgl. Slmb and PAR-2 act by different mechanisms, since the former is a subunit of the SCF ubiquitin ligase complex and promotes the degradation of the Par-6/aPKC complex, whereas the latter functions by recruiting PAR-1. Nevertheless, it is intriguing that PAR-2 contains a RING finger domain that is typically found in ubiquitin ligases, suggesting that it might have lost this activity during evolution (Morais-de-Sa, 2014).
The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. This study shows that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and Aurora Bkinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. It is proposed that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. These findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions (Bell, 2014).
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