Gene name - lethal (2) giant larvae
Synonyms - lgl; lethal giant larvae
Cytological map position - 21A3--4
Function - cytoskeletal component
Symbol - l(2)gl
FlyBase ID: FBgn0002121
Genetic map position - 2-0.0
Classification - WD40 repeats protein
Cellular location - cytoplasmic
|Recent literature||Calleja, M., Morata, G. and Casanova, J. (2016).
The tumorigenic properties of Drosophila
epithelial cells mutant for lethal giant larvae. Dev
Dyn [Epub ahead of print]. PubMed ID: 27239786
Mutations in Drosophila tumour suppressor genes (TSGs) lead to the formation of invasive tumours in the brain and imaginal discs. This study analyzed the tumorigenic properties of imaginal discs mutant for the TSG gene lethal giant larvae (lgl). lgl mutant cells display the characteristic features of mammalian tumour cells: they can proliferate indefinitely, induce additional tracheogenesis (an insect counterpart of vasculogenesis) and invade neighbouring tissues. Lgl mutant tissues exhibit high apoptotic levels, which lead to the activation of the Jun-N-Terminal Kinase (JNK) pathway. The study proposes that JNK is a key factor in the acquisition of these tumorigenic properties; it promotes cell proliferation and induces high levels of Mmp1 and confers tumour cells capacity to invade wildtype tissue. Noteworthy, lgl RNAi-mediated down regulation does not produce similar transformations in the CNS, thereby indicating a fundamental difference between the cells of developing imaginal discs and those of differentiated organs. The study discusses these results in the light of the "single big-hit origin" of some human paediatric or developmental cancers.
|Parsons, L. M., Grzeschik, N. A., Amaratunga, K., Burke, P., Quinn, L. M. and Richardson, H. E. (2017). A kinome RNAi screen in Drosophila identifies novel genes interacting with Lgl, aPKC and Crb cell polarity genes in epithelial tissues. G3 (Bethesda) 7(8):2497-2509. PubMed ID: 28611255
In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, a boutique collection of RNAi stocks targeting the kinome was screened for their capacity to modify Drosophila 'cell polarity' eye and wing phenotypes. Initially kinase or phosphatase genes were identified whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). Next a secondary screen was conducted to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, it was demonstrated that the 'nutrient sensing' kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, this screen has revealed novel cell-polarity interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.
|Gupta, R. P., Bajpai, A. and Sinha, P. (2017). Selector genes display tumor cooperation and inhibition in Drosophila epithelium in a developmental context-dependent manner. Biol Open 6(11): 1581-1591. PubMed ID: 29141951
During animal development, selector genes determine identities of body segments and those of individual organs. Selector genes are also misexpressed in cancers, although their contributions to tumor progression per se remain poorly understood. Using a model of cooperative tumorigenesis, this study shows that gain of selector genes results in tumor cooperation, but in only select developmental domains of the wing, haltere and eye-antennal imaginal discs of Drosophila larva. Thus, the field selector, Eyeless (Ey), and the segment selector, Ultrabithorax (Ubx), readily cooperate to bring about neoplastic transformation of cells displaying somatic loss of the tumor suppressor, Lgl, but in only those developmental domains that express the homeo-box protein, Homothorax (Hth), and/or the Zinc-finger protein, Teashirt (Tsh). In non-Hth/Tsh-expressing domains of these imaginal discs, however, gain of Ey in lgl- somatic clones induces neoplastic transformation in the distal wing disc and haltere, but not in the eye imaginal disc. Likewise, gain of Ubx in lgl- somatic clones induces transformation in the eye imaginal disc but not in its endogenous domain, namely, the haltere imaginal disc. These results reveal that selector genes could behave as tumor drivers or inhibitors depending on the tissue contexts of their gains.
|Daniel, S. G., Russ, A. D., Guthridge, K. M., Raina, A. I., Estes, P. S., Parsons, L. M., Richardson, H. E., Schroeder, J. A. and Zarnescu, D. C. (2018). miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila. Biol Open 7(1). PubMed ID: 29361610
Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, it was hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, it was found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling, a core set of ten miRNAs were identified that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which was found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, it was overexpressed in the context of lgl knock-down by RNAi, and it was found to be able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. This work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.
|Shu, Z., Huang, Y. C., Palmer, W. H., Tamori, Y., Xie, G., Wang, H., Liu, N. and Deng, W. M. (2017). Systematic analysis reveals tumor-enhancing and -suppressing microRNAs in Drosophila epithelial tumors. Oncotarget 8(65): 108825-108839. PubMed ID: 29312571
Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insights into how miRNAs contribute to primary tumor formation, an RNA sequencing (RNA-Seq) analysis was conducted of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (Ras(V12)), and 51 mature miRNAs were identified that changed significantly in tumorous discs. Followed by in vivo tumor enhancer and suppressor screens in sensitized genetic backgrounds, ten tumor-enhancing (TE) miRNAs and eleven tumor-suppressing (TS) miRNAs were identified that contributed to the nTSG defect-induced tumorigenesis. Among these, four TE and three TS miRNAs have human homologs. From this study, 29 miRNAs were identified that individually had no obvious role in enhancing or alleviating tumorigenesis despite their changed expression levels in nTSG tumors. This systematic analysis, which includes both RNA-Seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and functional relevance in epithelial tumorigenesis, whereas the evolutionarily conserved TE and TS miRNAs provide potential therapeutic targets for epithelial tumor treatment.
|Tseng, C. Y. and Hsu, H. J. (2017). Decreased expression of lethal giant larvae causes ovarian follicle cell outgrowth in the Drosophila Scutoid mutant. PLoS One 12(12): e0188917. PubMed ID: 29261681
Snail, a zinc-finger transcription factor, controls the process of epithelial-mesenchymal transition, and ectopic expression of this protein may produce cells with stem cell properties. Because the effect of Snail expression in ovarian epithelial cells remains unclear, this study generated Drosophila ovarian follicle stem cells (FSCs) with homozygous Scutoid (Sco) mutation. The Sco mutation is a reciprocal transposition that is known to induce ectopic Snail activity. Sco mutant FSCs showed excess proliferation and high competitiveness for niche occupancy, and the descendants of this lineage formed outgrowths that failed to enter the endocycle. Surprisingly, such phenotypes were not rescued by suppressing Snail expression, but were completely restored by supplying Lethal giant larvae (Lgl). The lgl allele is a cell polarity gene that is often mutated in the genome. Importantly, Sco mutants survived in a complementation test with lgl. This result was probably obtained because the Sco-associated lgl allele appears to diminish, but not ablate lgl expression. While these data do not rule out the possibility that the Sco mutation disrupts a regulator of lgl transcription, the results strongly suggest that the phenotypes found in Sco mutants are more closely associated with the lgl allele than ectopic Snail activity.
|Paul, M. S., Singh, A., Dutta, D., Mutsuddi, M. and Mukherjee, A. (2018). Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling. BMC Res Notes 11(1): 247. PubMed ID: 29661224
Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble, lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. This study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. Drosophila was used as an in vivo model to show the synergy between activated Notch (Nact) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of Nact and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. Activation of JNK signaling and upregulation of its receptor, Grindelwald in Nact /lgl-IR tumor. In contrast to previously described Nact/scrib-/- tumor, these experiments in Nact/lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.
Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. A group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Inactivation of the lethal(2)giant larvae gene results in malignant transformation of imaginal disc cells and neuroblasts of the larval brain in Drosophila. Subcellular localization of the l(2)gl gene product, P127, and its biochemical characterization have indicated that it participates in the formation of the cytoskeletal network. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic, imaginal disc and follicular epithelia. Mutation of tumor suppressor genes l(2)gl and discs-large (dlg) has identical effects on all three epithelia. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of l(2)gl gene product and junctional localization of Scrib and Dlg. scrib, dlg, and l(2)gl show strong genetic interactions. It is concluded that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control (Bilder, 2000b).
This cooperative interaction has been highlighted in two studies (Ohshiro, 2000 and Peng, 2000) showing the interaction of l(2)gl gene product and Dlg in the asymmetric cortical localization of all basal determinants in mitotic neuroblasts. In Drosophila, neuroblasts undergo typical asymmetric divisions to produce a progeny 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. Therefore, l(2)gl is indispensable for neural fate decisions. l(2)gl gene product, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Discs large participates in this process by regulating the localization of l(2)gl gene product. The localization of the apical components is unaffected in l(2)gl or dlg mutants. Dlg protein is apically enriched and is required for maintaining cortical localization of l(2)gl protein. Basal protein targeting requires microfilament and myosin function, yet the l(2)gl phenotype is strongly suppressed by reducing levels of myosin II (Zipper). Thus, l(2)gl gene product 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 and Peng, 2000).
Classically, genetic analysis has been used to reveal the biological function of l(2)gl. Genetic and phenotypic analyses have been performed of a temperature-sensitive mutation (l(2)glts3) that behaves as a hypomorphic allele at restrictive temperature. In experimentally overaged larvae obtained by using mutants in the production of ecdysone, the l(2)glts3 mutation displays a tumorous potential. This temperature-sensitive allele of the l(2)gl gene has been used to describe the primary function of the gene before tumor progression. A reduced contribution of both maternal and zygotic activities in l(2)glts3 homozygous mutant embryos blocks embryogenesis at the end of germ-band retraction. The mutant embryos are consequently affected in dorsal closure and head involution and show a hypertrophy of the midgut. These phenotypes are accompanied by an arrest of the cell shape changes normally occurring in lateral epidermis and in epithelial midgut cells. l(2)gl activity is also necessary for larval life and the critical period falls within the third instar larval stage. Finally, l(2)gl activity is required during oogenesis and mutations in the gene disorganize egg chambers and cause abnormalities in the shape of follicle cells, which are eventually internalized within the egg chamber. These results together with the tumoral phenotype of epithelial imaginal disc cells strongly suggest that the l(2)gl product is required in vivo in different types of epithelial cells to control their shape during development (Manfruelli, 1996).
Homozygous l(2)glts3 embryos from crosses between l(2)glts3/CyO females and homozygous l(2)glts3 males reared at 29°C develop into viable homozygous mutant larvae, most of them dying as early pupae. Strong reduction of the maternal contribution to l(2)gl expression by using homozygous l(2)glts3 females leads to an embryonic lethality of eggs deposited at 29°C. Reduction in maternal component can partially be complemented by introduction in the zygote of a wild-type copy of the gene. It can therefore be concluded that l(2)gl activity is required in embryogenesis. The maternal contribution to l(2)gl expression is sufficient for the embryogenesis to proceed normally and, in its absence, a partial zygotic rescue can be obtained, suggesting that the maternal and zygotic expressions are functionally equivalent. Eggs laid at 29°C by l(2)glts3 homozygous females or l(2)glts3/Df net62 females crossed with l(2)glts3 homozygous males were allowed to develop at 29°C and examined for embryonic defects resulting from a loss of function of l(2)gl. These embryos had a greatly reduced maternal component and were not rescued by the mutant zygote. In this situation, development of mutant embryos is blocked at germ band shortening. A typical mutant embryo is 'shrimp-shaped'. The involution of the head and the distal region of the embryos are also affected, but segmentation appears normal. The observed phenotype is almost 100% penetrant and indistinguishable for embryos of l(2)glts3/l(2)glts3 or l(2)glts3/Dfnet62 genotypes (Manfruelli, 1996).
During dorsal closure, cells of the lateral epidermis undergo a change in shape as the epidermal sheet spreads over the amnioserosa. The epidermal cells become thinner along the anteroposterior axis and longer along the dorsoventral axis. This change is completed at dorsal closure when all epidermal cells have acquired their final elongated shape. The movement is in fact initiated in the dorsal edge of epidermal cells and is later transmitted from cell to cell along the dorsoventral axis. The dramatic change in shape of ectodermal cells does not occur in a l(2)gl mutant. This phenotype is highly reminiscent of that of zipper mutants (zipper codes for cytoplasmic myosin II heavy chain) or of scab mutants (Manfruelli, 1996).
In addition to cell shape change in a wild-type embryo, there is a relocalization of cell junction proteins such as Fasciclin III. During dorsal closure, the protein present in the ectodermal cells of the dorsal edge is not initially expressed on the membrane facing the amnioserosa. When the two edges of the ectodermal cells join at dorsal midline, Fasciclin III becomes expressed uniformly at sites of cell-to-cell contacts in ectodermal cells of the dorsal edge as is usually the case in all other ectodermal cells. In l(2)gl mutants, ectodermal cells of the dorsal edge remain round and show, in many instances, an absence of polarity. As a consequence, Fasciclin III is expressed on the entire surface of these cells. Transmission of cell shape change to lateral and ventral epidermal cells is also affected in mutant embryos (Manfruelli, 1996).
Another major defect appears in embryos that have spent 12 hours or more at 29°C: they display hypertrophy of the midgut, which does not develop normally. Progression of intestinal hypertrophy during development was visualized with the aid of the l(2)gl cDNA which is still efficiently expressed in l(2)glts3 mutants. In late wild-type embryos, l(2)gl transcripts and P127 (Strand, 1994a) are mainly found in the midgut endodermal cells. In the mutant, midgut formation initiates correctly with the three constrictions appearing at the correct time even though germ band shortening and dorsal closure are blocked. However, instead of the lengthening and convolution of the midgut that normally occur in later stages, the mutant midgut enlarges to a size 2 to 3 times greater than that of the wild type, leading to a swelling of the embryos, especially in the dorsal part where the epidermis fails to form. In this process, the anterior lobe is systematically larger than the others. Semi-thin sections show that the yolk, which is digested at the end of embryogenesis in wild-type animals, is still present inside the intestine. These results suggest that the intestinal cells that are responsible for the hydrolysis of the yolk do not differentiate properly. A labelling with antibodies directed against Fasciclin III did not detect abnormalities in the formation of the visceral mesoderm of mutant embryos and this eliminates a possible cause for midgut epithelium malformation. It is noteworthy that, in other mutants, such as scab for example, which are also affected in dorsal closure, the midgut protrudes from the embryo, although shape and differentiation of intestinal cells appear normal with no sign of hypertrophy (Manfruelli, 1996).
The most conspicuous aspect of the mutant phenotype is a profound shape alteration of intestinal cells. Three successive changes take place in the shape of precursors of midgut epithelial cells. It has been assumed these modifications are related to a modulation of cell-to-cell adhesiveness during midgut development. Analysis of l(2)gl mutant phenotypes suggests that the two first steps, formation of an epithelium from mesenchymal precursor midgut cells and their flattening with reduced cellular adhesiveness, occurs correctly at stage 14. Later in embryogenesis, wild-type midgut epithelial cells become thinner and longer to constitute a tube-like and highly convoluted larval intestine. In l(2)gl mutants, this transformation does not occur and the epithelial cells seem to arrest their evolution at the previous stage. In several regions of the midgut, epithelial cells are rounder and flatter than the cells from a wild-type embryo of the same age. This defect could in part explain the apparent hypertrophy of the midgut, each cell occupying a larger area than in the wild-type embryo at the same stage (Manfruelli, 1996).
No attempt was made to check whether midgut epithelial cells continue to divide but, in l(2)gl mutant embryos, the midgut adult precursor cells appear to be in greater number and this overproliferation could also participate to some extent in the hypertrophied midgut phenotype (Manfruelli, 1996).
Temperature-sensitive alleles of the l(2)gl gene have been used to determine the sensitive period for embryonic lethality. In these experiments, it has been assumed that a temperature upshift produces an inactive l(2)gl protein, probably by a conformational change, and that this process is immediate and possibly reversible (Suzuki, 1970). Indeed, a protein whose size is indistinguishable from that of the wild-type component is produced by the l(2)glts3 mutant at 29°C. Shifts between permissive (22°C) and restrictive (29°C) temperature were applied to 1 hour egg-layings obtained from l(2)glts3 /l(2)glts3 flies. The gene activity is required very early in embryogenesis and the critical period extends from stage 8 to stage 12. Embryos that have spent 5 to 6 hours at 29°C (late stage 11, germ-band shortening) are no longer able to pursue their development, even if they are placed back at 22°C. Therefore some irreversible events have occurred during this period, indicating that l(2)gl is involved in some early stages of embryogenesis. By contrast, temperature upshifts performed after 10 hours of development do not have deleterious effects on development of homozygous l(2)glts3 embryos, which develop into larvae with apparently normal midguts; however, they die at the end of the third instar larval stage if maintained at 29°C. This result is somehow unexpected because, at this stage, l(2)gl is abundantly expressed in precursor cells of the epithelial midgut in which morphological defects have been shown to appear when the mutant embryos are maintained at 29°C from the time of fertilization (Manfruelli, 1996).
The hypomorphic character of the l(2)glts3 allele seems to be in part responsible for this behaviour. Upshifts experiments were performed on descendants of a cross between l(2)glts3/Dfnet62 females and homozygous l(2)glts3 males. In this case, the curve is systematically situated below that obtained in the case of a homozygous l(2)glts3 strain. A wide variety of phenotypes are observed depending both on the time at which the upshift is performed and the genotype of the embryos. The effect on germ-band shortening ranges from an extreme phenotype in the case of an early temperature upshift to an almost complete dorsal closure when the temperature upshift is applied later (Manfruelli, 1996).
In the same line, the effect of the l(2)glts3 mutation can be strengthened by performing temperature upshifts at 31°C. For example, more than 90% of the l(2)glts3 embryos show an arrest in dorsal closure when the temperature upshift is made at stage 11, shortly before the beginning of dorsal closure. By comparison, less than 50% of mutant embryos display a similar phenotype when temperature upshifts are carried out at 29°C. By contrast, the intestinal phenotype can only be observed in a small proportion of mutant embryos when the upshift at 31°C is made at early stage 15. This latter observation might suggest that the intestinal phenotype is not be a direct consequence of the loss of function of l(2)gl (Manfruelli, 1996).
However, due to the fact that l(2)glts3 is a hypomorphic allele at 29°C, a small proportion of the protein produced by the mutant could still be functional. In addition, the active conformation of the protein could be stabilized by its integration into a multicomponent complex (Strand, 1994b) which would then be difficult to displace once formed. The amount of l(2)glts3 protein recovered in an insoluble fraction, which is indicative of its complexed form (Strand, 1994b), was compared when different conditions of temperature upshifts were imposed on l(2)glts3. Mutant embryos persistently grown at 29°C contain no insoluble form of the P127 protein whereas embryos that have been submitted to the temperature upshift after 11-13 hours at 25°C contain a substantial proportion of this same insoluble protein, even though to a lesser extent than in wild-type embryos. This observation could explain why the late temperature upshifts did not produce the phenotypes that are routinely observed when the embryos are grown at non-permissive temperature. The complex formed at permissive temperature at early stages would be stable and functional and not able to be displaced when the temperature is raised (Manfruelli, 1996).
l(2)gl function is also required during larval life and the sensitivity period falls within the third instar larval stage. These data are in disagreement with previous experiments carried out using genetic mosaics that have shown that larval expression of l(2)gl is not required for the viability and hatching of the pupae (Merz, 1990). The different rationale underlying these two types of experiments could explain these opposite results. Two other observations lend support to this conclusion. By crossing a strain carrying a UAS-l(2)gl cDNA construct with a 69B-GAL4 strain in which the GAL4 transcription factor is specifically expressed in the precursors and the derivatives of the ectoderm layer, it has been possible to regulate, by temperature shifts, temporal l(2)gl expression in the progeny of this cross. The experiments were performed in a l(2)gl4 mutant background, which displays a null phenotype. The cross reared at 22°C, a temperature at which GAL4 is weakly active, never led to the recovery of l(2)gl4 homozygous viable adults. By contrast, at 29°C, GAL4 is fully active and consequently viable although sterile homozygous adults were recovered in the expected proportion. In this case, also, temperature-shift experiments allowed the determination of the same critical period for the l(2)gl gene activity. The weak activity of the GAL4 system at 22°C during embryogenesis and first larval stages could be sufficient to prevent the formation of tumors in late larvae. The same type of results were obtained with transgenic larvae carrying the l(2)gl cDNA under the control of a heat-shock promoter. Heat-shock delivered at the third instar larval stage is able to rescue a small percentage of adults. The absence of a tumoral phenotype in such animals reared at 22°C was also interpreted by a leaky expression of the heat-shock promoter sufficient to prevent the formation of tumors (Manfruelli, 1996).
The results suggest that a l(2)gl product is maternally provided to the embryo and that this expression is responsible for normal development of the homozygous mutant embryos at least until late larval stages. This implies expression of the l(2)gl gene during oogenesis and Szabad (1991) has shown that this expression is indeed required both in germ-line and follicle cells. The temperature-sensitive allele l(2)glts3 has been used to study the function of l(2)gl in oogenesis. The fertility of l(2)glts3 homozygous females maintained at 29°C progressively decreases, with a reduction in egg-laying of about 50% after 3 days and of 90%-95% after 7 days at this temperature. This effect is not observed in homozygous l(2)glts3 females carrying a transposon containing the Drosophila pseudoobscura gene which is (Torok, 1993) capable of rescuing the lethal phenotype associated to the conditional l(2)glts3 mutation (Manfruelli, 1996).
In examining an ovary from a homozygous mutant female grown at 29°C for 6 days, oogenesis appears to be blocked at stage 7-8. Older egg chambers have a necrotic aspect and do not develop normally. Egg chambers blocked at stages 7-8 all show the same phenotype, a multilayered accumulation of cells, probably of follicular origin, at their anterior and posterior tips. These cells have lost the correct polarity of follicular cells. The mutant cells are rounder than in the wild-type and, more interestingly, have internalized into the egg chamber within the space usually occupied by the ooplasm. Another phenotype, although not totally penetrant, shows a fusion of the germarium with the youngest egg chambers. This phenotype is better visualized by using the amorphic l(2)gl4 mutation. Overexpression of the l(2)gl cDNA in a homozygous l(2)gl4 background at third instar larval stage leads to recovery of a small proportion of viable adults. The females are however sterile. Their ovaries appear very small and the early egg-chamber fusion phenotype is observed in almost all the ovarioles. Under these conditions therefore, the same phenotype is obtained for the hypomorphic l(2)glts3 allele and the null l(2)gl4 allele carrying the transgene with, however, a better penetrance in the latter case (Manfruelli, 1996).
In spite of pleiotropic aspects of the mutations in l(2)gl, which are compatible with a rather ubiquitous expression of this gene product, the study presented here has uncovered some features shared by all phenotypes. (1) Mutations in l(2)gl essentially affect cells committed to an epithelial fate. (2) They apparently alter neither determination nor identity of embryonic cells but rather the differentiation state of epithelial cells. The process of shape remodeling that occurs in ectodermal cells during dorsal closure or in the midgut epithelial cells is abolished in l(2)gl mutants. An alteration of cell adhesiveness and loss of cell polarity is observed during oogenesis in follicle cells of the egg chambers, in tumorous imaginal discs (Gateff, 1978) and in salivary glands (Manfruelli, 1996).
The cytoskeleton has been extensively implicated in control of cell shape during Drosophila development. The results presented here and the fact that l(2)gl has been shown to be a cytoskeletal protein (Strand, 1994a), strongly suggest that l(2)gl functions during development as a regulator of cytoskeleton organization in epithelial cells. Genes whose mutations lead to analogous phenotypes are expected to act either separately or in cooperation in the same cellular process. Mutations in three other genes, zipper, coracle and scab hamper dorsal closure in a manner that is analogous although not identical to that prevailing in l(2)gl mutants, the phenotype in epidermis of scab mutants being the closest. The zipper gene encodes the cytoplasmic myosin heavy chain, which is considered a driving force for change in shape of the dorsal leading edge ectodermal cells. Furthermore, a direct molecular interaction between the non-muscle heavy chain and P127 has been observed (Strand, 1994b). The coracle gene codes for a protein associated with septate junctions homologous to the membrane-cytoskeleton protein 4.1 (Manfruelli, 1996 and references therein).
Two Drosophila genes coding for the Ras-related small GTP-binding proteins, DracA and DracB, homologous to mammalian Rac1 and Rac2, have been identified. Expression of transgenes bearing a dominant inhibitory version of the DracA cDNA under control of the hsp70 promoter causes a high frequency of defects in dorsal closure that are due to disruption of cell shape changes in lateral epidermis. These effects are associated with an altered localization of actin and myosin probably caused by cytoskeleton perturbations. P127 could be one of the components acting downstream of these Rho proteins and directly acting on the actin cytoskeleton and regulating the actin-myosin network necessary for cell-shape changes during epidermal development. P127 is found associated in a multicomponent complex containing one protein with protein kinase A activity (Strand, 1994b). It has been shown that protein kinase A may regulate microfilament integrity through phosphorylation and inhibition of the myosin light chain kinase activity in non-muscle cells and it could form a link between the cytoskeleton and the signal transduction regulating the actin-myosin pathway (Manfruelli, 1996 and references therein). Proximate components of this complex may include discs large, and scribbled, which act together with l(2)gl to properly localize apical proteins and adherens junctions to organize epithelial architecture (Bilder, 2000b).
These observations suggest that P127 might interact with these different gene products to generate a network connecting cytoskeleton and plasma membrane. Absence of the protein would result in a loosening of the network and eventually in a loss of cell adhesiveness and cell polarity. To support this hypothesis, experiments clearly demonstrating a direct interaction of the implicated proteins as well as a genetic interaction between the different genes should be performed. By delaying the puparation with the aid of an ecdysone temperature-sensitive mutation, it has been demonstrated that the hypomorphic l(2)glts3 allele has a tumoral potentiality. Double mutant ecdysoneless1/l(2)glts3 larvae never pupariate and can stay alive for 2-3 weeks. This delay could be the cause of the formation of tumors which then have had enough time to grow (Bryant, 1985). Alternatively, low ecdysone titer conditions could also be involved in tumor growth resulting from a lack of l(2)gl activity in l(2)gl larvae, as already suggested (Gateff, 1974). Another important conclusion is that l(2)gl activity is required during larval development to prevent the overgrowth phenotype (Manfruelli, 1996 and references therein).
Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).
Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).
The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl3A-GFP was expressed in the follicular epithelium. Lgl3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).
Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6S34A mutant cells. Temperature-sensitive aPKCts/aPKCk06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKCk06403) and kinase-defective (aPKCpsu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).
AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA37. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA37 mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA37 mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).
The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).
To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to LglWT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).
The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to LglS656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of LglS656A,S664A, mimicking the localization of Lgl3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of LglS656A,S660A and LglS660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).
RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).
Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).
Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl3A-GFP. More importantly, in contrast to LglS660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, LglS656A,S660A and LglS656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).
Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of LglS656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, LglS656A,S660A and LglS656A,S664A induce higher randomization of angles, whereas LglS660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).
Despite the ability of LglS656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of LglS656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that LglS656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in LglS656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).
In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).
It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).
The structure of the cDNAs of l(2)gl indicates the use of alternative splicing, either in the 5' untranslated exons or in the 3' coding exons. Thus the gene encodes two putative proteins of 1161 and 708 amino acids, p127 and p78, respectively, differing at their C termini. A 3'-truncated l(2)gl transposon that leaves the coding sequence of p78 intact but deletes 141 residues of p127 is capable of suppressing tumor formation in l(2)gl-deficient animals. These results suggest that the putative p78 protein is effective in controlling cell proliferation and/or differentiation (Jacob, 1987).
By structural, biochemical and molecular genetic analyses, the different mechanisms that control the expression of the lethal(2) giant larvae gene have been investigated. Transcription of the l(2)gl gene is controlled by two highly identical promoters that result from the duplication of the 2.8 kb proximal portion of the gene. These two repeats are 96% homologous. Reverse genetic analysis has shown that each promoter can drive gene expression. In addition to the promoters, both repeats express two or three exons according to the pattern of splicing. The most distal exon in the second repeat is required because it contains the ATG initiating codon at the beginning of the open reading frame. The 3' untranslated region appears to contain motifs that specifically destabilize the transcript. Deletion of this region results in the formation of more stable mRNAs. The l(2)gl gene is characterized by an unusual codon usage that may reflect an enhanced translation efficiency by moderating the strength of pairing between codons and anticodons and may therefore increase the expressivity of this gene (Strand, 1991).
A series of 32 chimaeric l(2)gl encoded 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. 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 (Jakobs, 1996).
date revised: 6 December 2000
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