lethal (2) giant larvae
Mutations in the tumor-suppressor gene lethal(2)giant larvae of Drosophila cause malignant transformation of the optic centers of the larval brain and the imaginal discs. The cloning and sequencing of the l(2)gl gene from Drosophila pseudoobscura is reported. Comparison of this sequence with D. melanogaster reveals a significant sequence conservation within the l(2)gl protein-coding domain and a strong sequence divergence in the 5' promoter region and in the introns. The deduced amino acid sequence of the D. pseudoobscura l(2)gl protein shows 17.7% divergence from D. melanogaster. However, despite these evolutionary differences, the D. pseudoobscura l(2)gl gene can fully suppress tumorigenicity and restore a normal development in l(2)gl-deficient D. melanogaster flies, although the rescued animals display poor viability and fertility. Furthermore, in D. melanogaster transgenic flies, the D. pseudoobscura l(2)gl protein is produced at a similar level as the D. melanogaster l(2)gl protein and displays an identical spatial pattern of expression. This shows that the highly divergent cis-regulatory elements of the D. pseudoobscura transgene can be fully recognized in D. melanogaster and lead to the synthesis of a transgenic protein that has enough specificity conserved for replacing the tumor-suppressor function normally fulfilled by the D. melanogaster l(2)gl protein (Torok, 1993).
By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids, was cloned. Cells deleted for SOP1 exhibit sensitivity to sodium stress, but show no sensitivity to general osmotic stress. Following exposure of sop1Delta cells to NaCl stress, the intracellular Na+ level and the Na+/K+ ratio rises to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produces only slight Na+ sensitivity. However, cells carrying a sop1Delta;sop2Delta double deletion become hypersensitive to Na+ and exhibit increased sensitivity also to Li+ and K+, suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p reveals a cytoplasmic distribution and cell fractionation studies show that a significant fraction of Sop1p is recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1Deltasop2Delta strain partially restores the Na+ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human (Larsson, 1997).
Inactivation of the tumor suppressor gene lethal(2) giant larvae of Drosophila leads to malignant transformation of the presumptive adult optic centers in the larval brain and tumors of the imaginal discs. These malignancies result from the disorganization of a cytoskeletal network in which the l(2)gl protein participates. A cDNA encoding the human homolog to the l(2)gl gene designated as hugl has been isolated. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, polyclonal rabbit antibodies have been prepared against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues (Strand, 1995).
The Drosophila melanogaster flightless-I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. The homologous human FLII gene encodes a 1269-residue protein with 58% amino acid sequence identity and is deleted in Smith-Magenis syndrome. The FLII gene has been cloned and its nucleotide sequence (14.1 kb) has been determined. FLII has 29 introns, compared with 13 in Caenorhabditis elegans and 3 in D. melanogaster. The positions of several introns are conserved in FLII-related genes and in the domains and subdomains of the gelsolin-like regions giving indications of gelsolin gene family evolution. In keeping with its function in indirect flight muscle in Drosophila, the human FLII gene was most highly expressed in muscle. The FLII gene lies adjacent to LLGL, the human homolog of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The 3' end of the FLII transcript overlaps the 3' end of the LLGL transcript, and the corresponding mouse genes Fliih and Llglh also overlap. The overlap region contains poly(A) signals for both genes and is strongly conserved between human and mouse (Campbell, 1997).
Drosophila lethal giant larvae (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and tumorigenesis in humans. The functional conservation between Drosophila lethal giant larvae and its human homologue Hugl-1(Llgl1) was investigated. Hugl-1 is lost in human solid malignancies, supporting its role as a tumour suppressor in humans. Hugl-1 expression in homozygous lgl Drosophila mutants is able to rescue larval lethality; imaginal tissues do not show any neoplastic features, with Dlg and Scrib exhibiting the correct localization; animals undergo a complete metamorphosis and hatch as viable adults. These data demonstrate that Hugl-1 can act as a tumour suppressor in Drosophila and thus is the functional homologue of lgl. Furthermore, the data suggest that the genetic pathway including the tumour suppressors lgl, dlg and scrib may be conserved in mammals, since human scrib and mammalian dlg can also rescue their respective Drosophila mutations. These results highlight the usefulness of fruit fly as a model system for investigating in vivo the mechanisms linking loss of cell polarity and cell proliferation control in human cancers (Grifoni, 2004).
The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, tumour tissues were evaluated from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and these findings were compared with the clinical data from each of these patients. Hugl-1 was found to be lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increases cell adhesion and decreases cell migration. These studies thus indicate that downregulation of Hugl-1 contributes to CRC progression (Schimanski, 2005).
It is speculated that the human homolog of Drosophila Lgl, Hugl-1, might play a role in epithelial-mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Melanoma cell lines and tissue samples of malignant melanoma were evaluated for loss of Hugl-1 transcription. Hugl-1 was found to be downregulated or lost in all cell lines and in most of the tumor samples analysed, and these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. These studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression (Kuphal, 2005).
Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p (closest Drosophila homolog CG17762) and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae. sro7Delta sro77Delta double mutants show a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of l(2)gl could suppress the sro7Delta sro77Delta defect. Similar to the l(2)gl protein, Sro7p forms a complex with Myo1p, a type II myosin. These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein. Genetic analysis revealed that deletion of SRO7 and SRO77 shows reciprocal suppression with deletion of MYO1 (i.e., the sro7Delta sro77Delta defect is suppressed by myo1Delta and vice versa). In addition, SRO7 shows genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppresses a defect in MYO2 and, conversely, overexpression of MYO2 suppresses the cold-sensitive phenotype of sro7Delta sro77Delta mutants. These results indicate that Sro7 function is closely related to both Myo1p and Myo2p. A model is proposed in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways (Kagami, 1998).
Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. A novel syntaxin-1-binding protein, tomosyn, has been identified that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation (Fujita, 1998).
A neural tissue-specific syntaxin-1-binding protein, tomosyn, is capable of dissociating Munc18/n-Sec1/rbSec1 from syntaxin-1 to form a 10S tomosyn complex, an intermediate complex converted to the 7S SNARE complex. Two splicing variants of tomosyn have been isolated: one has 36 amino acids (aa) insertion and another has 17 aa deletion. The original one has been named m-tomosyn, the big one b-tomosyn, and the small one s-tomosyn. s-Tomosyn as well as m-tomosyn is mainly expressed in brain whereas b-tomosyn is ubiquitously expressed. All the isoforms bind to syntaxin-1, but not to syntaxin-2, -3, or -4, and have a region highly homologous to VAMP, another syntaxin-binding protein. This region is necessary but not sufficient for high-affinity binding of tomosyn to syntaxin-1 (Yokoyama, 1999).
Two related yeast proteins, Sro7p and Sro77p, have been identified based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, Lethal (2) giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions since loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. Similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, no defect is seen in actin polarity under conditions where a dramatic effect is seen on secretion. This demonstrates that the primary function of Sro7/77 is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, these studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane (Lehman, 1999).
The Drosophila tumor suppressor protein Lethal (2) giant larvae [L(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. A mammalian homolog of L(2)gl, called Mlgl, has been characterized in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates coimmunoprecipitates with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis (Musch, 2002).
Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. Mammalian Lgl is shown to compete for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with a previous observation that the complex containing PAR-3 promotes it.These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity (Yamanaka, 2003).
Thus evidence is presented showing that the PAR-6β/aPKCλ complex interacts with either mLgl or PAR-3 in a mutually exclusive manner, forming two independent protein complexes. Notably, overexpression of mLgl-2 inhibits TJ formation; this finding is in direct contrast with the data found for PAR-3, whose overexpression, but not that of its mutant lacking the aPKC binding region, promotes TJ formation. This suggests that the two independent complexes have distinct functions in the establishment of epithelial cell polarity. This is consistent with the results of genetic studies of Drosophila in which Lgl is required for formation of the basolateral membrane domain through the inhibition of the formation of apical identity, whereas subapical Bazooka (PAR-3) is required for the formation of the apical membrane domain (Yamanaka, 2003).
In polarized epithelial cells, mLgl localizes to the lateral region, in contrast to the PAR-6β/aPKCλ/PAR-3 complex that localizes to the apical end of the lateral domain. Interestingly, mLgl-2 transiently codistributes with PAR-6β and aPKCλ during the initial phase of epithelial cell polarization, whereas PAR-3 stably codistributes with them at the apical end of the cell-cell contact region; this finding indicates that the balance between the two independent complexes changes during the initial phase of epithelial cell polarization. Further, overexpression of aPKCλ kn (kinase deficient aPKC) results in the abnormal codistribution of PAR-6β and mLgl-2 at the cell periphery; this finding suggests that aPKCλ activity is required for the segregation of PAR-6β and mLgl-2 localization during this process. Thus, the present results, as well as previous findings, led to the following working model. The cell-cell contact initially stimulates the localization of the protein complex containing PAR-6β, aPKCλ, and mLgl at the cell-cell contact region. The complex is 'inactive' for TJ formation. Once aPKCλ is activated, mLgl segregates from the PAR-6β/aPKCλ complex. This triggers the formation of the 'active' PAR-6β/aPKCλ/PAR-3 complex that promotes the formation of the epithelial junctional complex. Segregated mLgl remains in the lateral region and seems to contribute to the establishment of the basolateral membrane identity, because mLgl-1 has been reported to interact with syntaxin-4, a component of the basolateral exocytic machinery. Although the mechanism for activation of aPKCλ remains to be clarified, Cdc42 and/or Rac1 are strong candidates as activators of aPKC in MDCK cells, since the GTP-bound form of Cdc42 activates aPKCλ kinase activity through PAR-6 in vitro and cell-cell adhesion activates Cdc42 and Rac1 in epithelial cells (Yamanaka, 2003).
mLgl is phosphorylated by aPKCλ and this phosphorylation increases in response to cell-cell adhesion-mediated cell polarization. Further, a phosphomimicking mutant of mLgl-2 (3SE) fails to bind to aPKCλ. These results imply that aPKCλ-dependent phosphorylation of mLgl is involved in the regulation of its interaction with the PAR-6β/aPKCλ complex. In contrast, no difference could be detected between mLgl-2 wild-type and its 3SE mutant in their interactions with the PDZ domain of PAR-6β. In addition, overexpression of mLgl-2 mutants (3SA or 3SE) affects TJ formation similarly to that of wild-type. These results suggest the existence of another mechanism regulating the interaction of mLgl-2 with PAR-6β. Mammalian Crumbs/Stardust (Pals1) has been shown to interact with the PDZ domain of PAR-6β and this interaction is enhanced by activated Cdc42. Taken together with the present results, this suggests that the Crumbs/Pals1 complex might also be involved in the regulation of the interaction between mLgl and the PAR-6β/aPKCλ complex; the PAR-6β/aPKCλ complex, together with PAR-3, may involve the Crumbs/Pals1 complex to promote TJ formation. Thus, the dissociation of mLgl from the PAR-6β/aPKCλ complex likely triggers the interaction of the PAR-6β/aPKCλ complex with the Crumbs/Pals1 complex in addition to its interaction with PAR-3. The functional interactions proposed by this model are consistent with the results of recent genetic studies of Drosophila in which Lgl and Crumbs compete with each other to define respective membrane identity (Yamanaka, 2003).
The findings in this study suggest a notable analogy in the mechanism regulating epithelial polarity between Drosophila and mammals. This notion is supported by a recent observation in Drosophila that Lgl interacts with PAR-6 and aPKC and regulates the apicobasal polarity of Drosophila neuroblasts. In mammalian fibroblasts, mLgl-1 has been reported to form a protein complex with PAR-6α (also called PAR-6C) and aPKC and is involved in the polarized migration of wounded MEF cells. Further studies will further an understanding of the molecular mechanism underlying the establishment of cell polarity in a variety of biological contexts (Yamanaka, 2003).
Thus mammalian Lgl competes for PAR-3 in forming an independent protein complex with PAR-6 and aPKC in epithelial cells. During epithelial cell polarization, mLgl transiently colocalizes with PAR-6 and aPKC at the cell-cell contact region, and increased localization of mLgl and PAR-6 to the cell-cell contact region suppresses TJ formation. This finding contrasts with the data found for PAR-3, which promotes TJ formation and thus indicates that the balance between the two independent protein complexes regulates the establishment of epithelial cell polarity. It is also suggested that aPKC activity-mediated phosphorylation of mLgl is involved in the regulation of mLgl's interaction with PAR-6/aPKC. These findings provide new insight into the mechanism underlying the establishment of epithelial cell polarity (Yamanaka, 2003).
Disruption of cell polarity is seen in many cancers; however, it is generally considered a late event in tumor progression. Lethal giant larvae (Lgl) has been implicated in maintenance of cell polarity in Drosophila and cultured mammalian cells. Loss of Lgl1 in mice results in formation of neuroepithelial rosette-like structures, similar to the neuroblastic rosettes in human primitive neuroectodermal tumors. The newborn Lgl1-/- pups develop severe hydrocephalus and die neonatally. A large proportion of Lgl1-/- neural progenitor cells fail to exit the cell cycle and differentiate, and, instead, continue to proliferate and die by apoptosis. Dividing Lgl1-/- cells are unable to asymmetrically localize the Notch inhibitor Numb, and the resulting failure of asymmetric cell divisions may be responsible for the hyperproliferation and the lack of differentiation. These results reveal a critical role for mammalian Lgl1 in regulating of proliferation, differentiation, and tissue organization and demonstrate a potential causative role of disruption of cell polarity in neoplastic transformation of neuroepithelial cells (Klezovitch, 2004).
In early vertebrate development, apicobasally polarised blastomeres divide to produce inner non-polarised cells and outer polarised cells that follow different fates. How the polarity of these early blastomeres is established is not known. The role of Crumbs3, Lgl2 and the apical aPKC in the polarisation of frog blastomeres was examined. Lgl2 localises to the basolateral membrane of blastomeres, while Crumbs3 localises to the apical and basolateral membranes. Overexpression aPKC and Crumbs3 expands the apical domain at the expense of the basolateral and repositions tight junctions in the new apical-basolateral interface. Loss of aPKC function causes loss of apical markers and redirects basolateral markers ectopically to the apical membrane. Cell polarity and tight junctions, but not cell adhesion, are lost and outer polarised cells become inner-like apolar cells. Overexpression of Xenopus Lgl2 phenocopies the aPKC knockout, suggesting that Lgl2 and aPKC act antagonistically. This was confirmed by showing that aPKC and Lgl2 can inhibit the localisation of each other and that Lgl2 rescues the apicalisation caused by aPKC. It is concluded that an instrumental antagonistic interaction between aPKC and Lgl2 defines apicobasal polarity in early vertebrate development (Chalmers, 2005).
Cell polarity plays a critical role in the development of all metazoans; however, the mechanisms of cell polarity and the specific role of cell polarity pathways in mammalian organisms are still poorly understood. Lethal giant larvae (Lgl) is an apical-basal polarity gene identified in Drosophila, where it functions as a tumor suppressor controlling self-renewal and differentiation of progenitor cells. There are two orthologs of Lgl in mammalian genomes: Llgl1 and Llgl2. While mammalian Lgls are assumed to be tumor suppressor genes, little is known about their function in vivo. This study reports the functional analysis of murine Llgl2. Llgl2-/- mice were generated, and Llgl2 was found to function as a polarity protein required for proper branching morphogenesis during placental development. Llgl2-/- pups were found to be born as runts but quickly catch up in size and grow into normal-size adults. Surprisingly, no prominent phenotypes or spontaneous tumors were observed in adult Llgl2-/- mice. Analyses of placental trophoblasts reveal a critical role for Llgl2 in cell polarization and polarized cell invasion. It is concluded that mammalian Llgl2 is required for proper polarized invasion of trophoblasts and efficient branching morphogenesis during placental development, but, unlike its Drosophila ortholog, it does not function as a canonical tumor suppressor gene (Sripathy, 2011).
Epithelial cells are equipped with junctional complexes that are involved in maintaining tissue architecture, providing mechanical integrity and suppressing tumour formation as well as invasiveness. A strict spatial segregation of these junctional complexes leads to the polarisation of epithelial cells. In vertebrate epithelia, basally localised hemidesmosomes mediate stable adhesion between epithelial cells and the underlying basement membrane. Although components of hemidesmosomes are relatively well known, the molecular machinery involved in governing the formation of these robust junctions, remains elusive. This study has identified the first component of this machinery using a forward genetic approach in zebrafish; the function of penner (pen)/lethal giant larvae 2 (lgl2) is necessary for hemidesmosome formation and maintenance of the tissue integrity in the developing basal epidermis. Moreover, in pen/lgl2 mutant, basal epidermal cells hyper-proliferate and migrate to ectopic positions. Of the two vertebrate orthologues of the Drosophila tumour suppressor gene lethal giant larvae, the function of lgl2 in vertebrate development and organogenesis have remained unclear. This study unravels an essential function of lgl2 during development of the epidermis in vertebrates (Sonawane, 2005).
Vertebrates have two orthologues of the Drosophila gene lethal giant larvae. Of these, lgl1 knockout mice exhibit brain dysplasia without defects in any other tissues. In these mice, components of the apical junctional complex such as ß-catenin, myosin II-B and f-actin are disorganised, indicating loss of polarity in the neuroepithelium. Analyses in zebrafish have revealed that pen/lgl2 functions in the formation of basally localised hemidesmosomes and maintenance of the basal localisation of keratin cytoskeleton. The localisation of actin and ß-catenin at lateral and apical borders of basal cells remains unaffected before and after the onset of hemidesmosomal phenotype. Some decrease is observed in the intensity of ß-catenin staining; cells express hemidesmosomal phenotype but this phenotype does not seem to be the primary effect of the mutation because even clones carrying lgl2MO, which would also knockdown maternal lgl2 expression, did not show any phenotype on 3.5 dpf that was indicative of loss of ß-catenin at the apical or lateral borders. Thus, lgl2 is primarily involved in hemidesmosome formation, a process that is involved in the maturation of the basal domain during epidermal development. Involvement of lgl1 in maintenance of apical junctional complex in the brain and that of lgl2 in the formation of hemidesmosomes and maintenance of the cellular morphology in basal epidermal cells indicate that these two Drosophila lgl orthologues may have evolved tissue specific functions during vertebrate development. This statement is further supported by the fact that lgl1 transcripts are absent in developing epithelia wherein lgl2 transcripts are abundant (Sonawane, 2005).
The integrity and homeostasis of the vertebrate epidermis depend on various cellular junctions. How these junctions are assembled during development and how their number is regulated remain largely unclear. This study addressed these issues by analysing the function of Lgl2, E-cadherin and atypical Protein kinase C (aPKC) in the formation of hemidesmosomes in the developing basal epidermis of zebrafish larvae. It has been shown that a mutation in lgl2 (penner) prevents the formation of hemidesmosomes. This study shows that Lgl2 function is essential for mediating the targeting of Integrin alpha 6 (Itga6), a hemidesmosomal component, to the plasma membrane of basal epidermal cells. In addition, it was shown that whereas aPKClambda seems dispensable for the localisation of Itga6 during hemidesmosome formation, knockdown of E-cadherin function leads to an Lgl2-dependent increase in the localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to control the localisation of Itga6 during the formation of hemidesmosomes in the developing epidermis (Sonawane, 2009).
How do Lgl2 and E-cadherin, localised at the lateral domain, regulate the formation of hemidesmosomes formed at the basal domain in epidermal cells? It was shown at the lateral domain, Itga6 localises with Lgl2 as well as with E-cadherin. This observation indicates that after its synthesis, a fraction of Itga6 is first targeted to the lateral domain. This lateral Itga6 fraction diminishes by 5 days of development, indicating that Itga6 localisation at the lateral domain is dynamic. In early lgl2 mutant larvae (3.75 days), there is a selective loss of Itga6 localisation at the lateral membrane domain. Moreover, in lgl2 mutant larvae, Itga6 vesicles accumulate in the cytoplasm, especially near the lateral and apical domains. Thus, it is plausible that beyond 3.5 days, a fraction of the Itga6 synthesised is targeted to the lateral membrane domain first and that Lgl2 mediates this targeting. This fraction at the lateral domain then translocates to the basal domain, where it joins the existing Itga6 fraction (localised prior to 3.5 days) clustered at the intermediate filaments, and becomes assembled into functional hemidesmosomes. The translocation of the lateral Itga6 fraction to the basal domain may occur by passive diffusion or by transcytosis. In the latter case, a likely mechanism might be Rab21/Rab5-mediated endocytosis and trafficking of Itga6 from the lateral domain and Rab11-mediated delivery to the basal domain through recycling endosomes. Since, in lgl2 mutant larvae, the Itga6 fraction targeted beyond 3.5 dpf fails to reach the lateral membrane domain and thus also the basal domain, the existing levels of Itga6 at the basal domain (localised before 3.5 dpf) remain insufficient to form functional hemidesmosomes (Sonawane, 2009).
During neurogenesis in Xenopus, apicobasally polarised superficial and non-polar deep cells take up different fates: deep cells become primary neurons while superficial cells stay as progenitors. It is not known whether the proteins that affect cell polarity also affect cell fate and how membrane polarity information may be transmitted to the nucleus. This study examined the role of the polarity components, apically enriched aPKC and basolateral Lgl2, in primary neurogenesis. A membrane-tethered form of aPKC (aPKC-CAAX) suppresses primary neurogenesis and promotes cell proliferation. Unexpectedly, both endogenous aPKC and aPKC-CAAX show some nuclear localisation. A constitutively active aPKC fused to a nuclear localisation signal has the same phenotypic effect as aPKC-CAAX in that it suppresses neurogenesis and enhances proliferation. Conversely, inhibiting endogenous aPKC with a dominant-negative form that is restricted to the nucleus enhances primary neurogenesis. These observations suggest that aPKC has a function in the nucleus that is important for cell fate specification during primary neurogenesis. In a complementary experiment, overexpressing basolateral Lgl2 causes depolarisation and internalisation of superficial cells, which form ectopic neurons when supplemented with a proneural factor. These findings suggest that both aPKC and Lgl2 affect cell fate, but that aPKC is a nuclear determinant itself that might shuttle from the membrane to the nucleus to control cell proliferation and fate; loss of epithelial cell polarity by Lgl2 overexpression changes the position of the cells and is permissive for a change in cell fate (Sabherwal, 2009).
Directed membrane trafficking is believed to be crucial for axon development during neuronal morphogenesis. However, the underlying mechanisms are poorly understood. This study reports a role of Lgl1, the mammalian homolog of Drosophila tumor suppressor Lethal giant larvae, in controlling membrane trafficking underlying axonal growth. Lgl1 was found to associated with plasmalemmal precursor vesicles and enriched in developing axons. Lgl1 upregulation promoted axonal growth, whereas downregulation attenuated it as well as directional membrane insertion. Interestingly, Lgl1 interacted with and activated Rab10, a small GTPase that mediates membrane protein trafficking, by releasing GDP dissociation inhibitor (GDI) from Rab10. Furthermore, Rab10 lies downstream of Lgl1 in axon development and directional membrane insertion. Finally, both Lgl1 and Rab10 are required for neocortical neuronal polarization in vivo. Thus, the Lgl1 regulation of Rab10 stimulates the trafficking of membrane precursor vesicles, whose fusion with the plasmalemma is crucial for axonal growth (Wang, 2011).
How might Lgl1 itself be regulated during neuronal polarization? A recent report showed that the mammalian homologs of yeast exocyst complex influence neuronal polarity through aPKC, which is known to regulate Lgl homologs in other systems. Furthermore, Lgl1 has been shown to be regulated by Disheveled (Dvl) (Dollar, 2005). It is thus possible that Dvl/aPKC may act as upstream regulators of the Lgl1-Rab10 system. It has been shown that vesicle-SNARE proteins, such as VAMP2, VAMP4, or VAMP7, are involved in the exocytic machinery that drives neuritogenesis. However, this study found that VAMP2 was not associated with Lgl1-coated beads, and downregulation of Lgl1 or Rab10 had no effect on membrane localization of VAMP2. Nevertheless, these results cannot exclude the possible interaction between the Lgl1-Rab10 system and other SNARE proteins (Wang, 2011).
The establishment of polarity in many cell types depends on Lgl, the tumour suppressor product of lethal giant larvae, which is involved in basolateral protein targeting. The conserved complex of Par3, Par6 and atypical protein kinase C phosphorylates and inactivates Lgl at the apical surface; however, the signalling mechanisms that coordinate cell polarization in development are not well defined. This study shows that a vertebrate homologue of Lgl associates with Dishevelled, an essential mediator of Wnt signalling, and Dishevelled regulates the localization of Lgl in Xenopus ectoderm and Drosophila follicular epithelium. Both Lgl and Dsh are required for normal apical-basal polarity of Xenopus ectodermal cells. In addition, the Wnt receptor Frizzled 8, but not Frizzled 7, causes Lgl to dissociate from the cortex with the concomitant loss of its activity in vivo. These findings suggest a molecular basis for the regulation of cell polarity by Frizzled and Dishevelled (Dollar, 2005).
In multicellular organisms polarity is a fundamental property of cells, one that is required for asymmetric division, changes in cell shape, adhesion and migratory behaviour. Recent studies have indicated that the core mechanisms of cell polarization are conserved. In Drosophila epithelial cells, sensory organ precursors and neuroblasts, as well as in mammalian cells, the apical Par (partitioning defective) complex, which consists of the PDZ-domain-containing proteins Par6 and Par3, and atypical protein kinase C (aPKC), regulates apical-basal polarity and proper localization of cell fate determinants. Activation of aPKC in the Par6 complex results in the phosphorylation of Lgl and its dissociation from the cortex. In neuronal precursors, Lgl is required for the asymmetric targeting of cell fate determinants. Lgl binds Syntaxin 4, a component of the basolateral exocytic machinery, and the yeast Lgl homologues Sro7 and Sro77 are required for polarized exocytosis. These findings support the view that Lgl controls cell polarity through basolateral targeting (Dollar, 2005).
In developing tissues, individual cells, although capable of intrinsic polarization, must polarize correctly in the context of their environment by responding to extracellular polarizing cues. In fact, both coordinated polarization of cells in the plane of epithelial tissue and the alignment of intercalating cells during vertebrate gastrulation require Frizzled (Fz) and Dishevelled (Dsh), which are components of the Wnt signalling pathway. In addition, members of the Wnt pathway have been implicated in many processes that also involve the Par6-Par3-aPKC pathway, including asymmetric division of Drosophila sensory organ precursors and mitotic spindle orientation in Caenorhabditis elegans blastomeres. Despite these functional similarities, an understanding of the biochemical communication between these two pathways remains limited (Dollar, 2005).
This study has identified a Xenopus homologue of Lgl as a protein that interacts with Dsh in a yeast two-hybrid screen, raising the possibility that Lgl is regulated by the Wnt pathway. To assay Lgl activity, the effect of overexpressing Lgl in Xenopus embryonic ectoderm was determined. Animal pole blastomeres express Lgl and have a well-defined apical-basal polarity, including the apical restriction of aPKC, a negative regulator of Lgl, suggesting that they are an appropriate in vivo system in which to examine Lgl activity. A full-length cDNA encoding Xenopus Lgl was obtained and expressed as a fusion protein with green fluorescent protein (GFP) or Myc in Xenopus embryos. Just before gastrulation, a marked change in pigment distribution was observed in superficial ectoderm cells overexpressing Lgl1. This phenotype was observed in most embryos injected with either GFP-Lgl1 or Myc-Lgl1 RNA and was dependent on dose, because the pigment redistribution became more evident when larger amounts of RNA were injected (Dollar, 2005).
To see whether these pigment changes reflected cell polarity defects, embryos expressing GFP-Lgl1 were cross-sectioned and double immunostained for GFP and either aPKC, an apical marker, or occludin, which marks the basolateral membrane and developing tight junctions in stage 10 embryos. Cells expressing GFP-Lgl1 showed a marked change in polarity as compared with uninjected cells, as judged by the loss of apical aPKC and the spread of occludin to the apical surface. Most GFP-Lgl1 was localized to the basolateral membrane, consistent with conserved regulation by endogenous aPKC. Some GFP-Lgl1 was detected at the apical surface, however, suggesting that the observed effects were due to ectopic Lgl1 activity (Dollar, 2005).
To determine the localization of endogenous Lgl1 in these cells, antibodies were raised against Xenopus Lgl1, which showed that it was restricted to the basolateral membrane. In addition, an Lgl1 construct in which conserved aPKC phosphorylation sites were mutated localized mostly to the apical surface and caused identical pigmentation and polarity changes when smaller amounts of RNA were injected. These and other observations indicate that the overexpression phenotype may result from ectopic Lgl1 activity at the apical surface. This interpretation is consistent with the ability of a mouse homologue of Lgl to inhibit tight junction formation in tissue culture cells (Dollar, 2005).
To confirm that Lgl and Dsh interact in vivo in Xenopus embryonic cells, immunoprecipitations were carried out with lysates from embryos expressing tagged proteins. The fragment of Lgl identified in the yeast screen (HA-LglC) specifically co-precipitated with full-length Myc-Xdsh. Similarly, in a complementary experiment full-length GFP-Lgl1 co-precipitated with a region of Xdsh containing the DIX domain (Xdsh-N). In addition, endogenous Dsh associated with both Lgl constructs. Because most Lgl1 and Dsh molecules do not colocalize at their steady-state levels in the cell, the two proteins are likely to interact transiently, reminiscent of the regulation of Lgl by Par6-aPKC (Dollar, 2005).
To evaluate whether Dsh is required for Lgl function, the activity of GFP-Lgl1 was examined in embryos injected with a morpholino antisense oligonucleotide (XdshMO) that has been shown to reduce specifically endogenous Xdsh. Depletion of Xdsh completely suppressed ectopic pigmentation caused by Lgl1 RNA, indicating that Dsh is required for Lgl1 activity. Consistent with this, an Lgl1 construct lacking the region involved in Dsh binding did not cause the pigmentation changes characteristic of full-length Lgl1, although it was expressed in similar amounts (Dollar, 2005).
Whether Dsh is required for the subcellular localization of Lgl was examined. The membrane localization of GFP-Lgl1 was abolished in embryos injected with XdshMO. Western blot analysis showed that Myc-Lgl1 protein was decreased in embryos depleted of Dsh in a dose-dependent manner. This decrease was specific to Lgl1, because XdshMO did not alter the amount of GFP, which was coexpressed in the same embryos as an internal control. In addition, XdshMO did not have the same effect on the expression of Myc-LglDeltaC. In a complementary experiment, overexpression of Dsh RNA in blastula ectoderm resulted in an increase in endogenous Lgl1. These observations indicate that Dsh may be involved in regulating the localization and stability of Lgl. It was confirmed that Dsh is required for localization of Lgl1 to the membrane by examining the distribution of endogenous Lgl1 in XdshMO-injected cells, using GFP as a lineage tracer. Almost no basolateral Lgl1 was detected in GFP-positive cells that received XdshMO. It is concluded that Dsh is required for Lgl1 localization and activity in vivo, possibly by stabilizing Lgl (Dollar, 2005).
To assess whether the regulation of Lgl by Dsh is conserved in other organisms, the distribution of Lgl was examined in dsh-null mutant clones in Drosophila. To avoid a possible compensatory effect of maternal Dsh, the requirement for Dsh was tested in the follicle cell epithelium, which forms around the oocyte relatively late in development. In follicle cells, Lgl is localized to the lateral membrane and in the nucleus. Mutant dsh follicle cell clones were generated with the FLP/FRT system. It was found that Lgl was delocalized from the membrane of dsh mutant cells. These results extend the observations made in Xenopus ectoderm to the fly follicular epithelium and show that Dsh has a conserved role in subcellular localization of Lgl (Dollar, 2005).
Next whether Lgl and Dsh are required for apical-basal polarity in embryonic ectoderm was tested, because the findings predicted that depletion of Lgl1 and Dsh would cause similar apical-basal polarity defects. A morpholino oligonucleotide was designed that specifically decreased Lgl1 protein in vivo by gastrulation stages (LglMO). Embryos injected with LglMO or XdshMO, together with GFP RNA as a lineage tracer, were sectioned and stained with antibodies to aPKC, occludin, beta-catenin and beta1-integrin, along with antibodies against GFP. Both Lgl1 and Dsh depletion resulted in a loss of apical aPKC and in ectopic accumulation of occludin at the apical surface. By contrast, the basolateral localization of beta-catenin and beta1-integrin was unaffected by Lgl and Dsh depletion. These results show that Lgl1 is required for some, but not all, aspects of apical-basal polarity of the ectoderm. The similar defects seen in Dsh-depleted cells are probably due to the role of Dsh in Lgl regulation. Most importantly, these findings establish that Dsh, as well as Lgl1, is involved in apical-basal epithelial polarization (Dollar, 2005).
Because Dsh is an essential mediator of Fz signalling, whether Fz signals can influence Lgl activity was examined. Overexpression of Fz receptors has been shown to mimic aspects of active signalling. Fz8, Fz7 and Fz3 RNAs were co-injected with Myc-Lgl1 or GFP-Lgl1 RNA into animal blastomeres of four-cell Xenopus embryos. It was found that Fz8, but not Fz7 or Fz3, RNA completely suppressed Lgl1-dependent pigment redistribution, indicating that Fz8 specifically interferes with Lgl function. Moreover, the membrane localization of GFP-Lgl1 was markedly altered by coexpression of Fz8, but not by Fz7. All three Fz RNAs were equally efficient in interfering with morphogenetic movements in later development, consistent with similar amounts of protein expression. Overall amounts of Myc-Lgl1 were slightly reduced by coexpression of Fz8, but not Fz7. In contrast to Dsh-depleted embryos, however, Myc-Lgl1 was still highly expressed in these embryos, indicating that Fz8 primarily affects Lgl localization (Dollar, 2005).
Lastly, the delocalization of Lgl in response to Fz8 was not limited to overexpressed Lgl, but was also observed for endogenous Lgl. Different Fz receptors seem to function in a compartmentalized manner in the cell, as suggested by their differential distribution along the apical-basal axis of Drosophila epithelial cells. This raises the possibility that in the assay Fz8 is acting in a dominant-negative manner by sequestering Dsh from its required subcellular localization. It was found that Fz8, as well as Fz7 and Fz3, recruits Dsh-GFP to the basolateral surface, however, supporting the notion that the effect of Fz8 on Lgl is receptor-specific. It was concluded that individual Fz receptors can differentially influence the intracellular distribution and activity of Lgl (Dollar, 2005).
The control of Lgl by Dsh suggests a general mechanism for the coordinated regulation of cell polarity by extracellular signalling that is based on existing apical-basal polarity cues. For example, localized Fz-Dsh signalling could provide positional cues for the Par3-Par6-aPKC complex by locally depleting Lgl, which acts antagonistically in the formation of this complex. Consistent with this model, in Drosophila embryos Fz and Dsh colocalize with the apical Par complex in ectoderm cells and restrict the Par complex to the posterior cortex in sensory organ precursors. A partial disruption of apical-basal polarity in the plane of epithelial tissue by localized Fz-Dsh signalling could contribute to planar cell polarity, whereas a more complete disruption could result in epithelial-mesenchymal transformation and altered cell movements that are crucial for morphogenesis. Further studies are warranted to examine the interactions between the molecules involved in regulating planar and apical-basal polarity and those controlling morphogenetic movements in development (Dollar, 2005).
The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. This study shows direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). Lgl1 and NMII-A form a complex in vivo, and data is provided that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, it was shown that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly (Dahan, 2012).
Non-muscle myosin IIA (NMII-A) and the tumor suppressor Lgl1 play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. This study shows that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition on NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. It was further shown that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the complexes formation is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, it was shown that aPKCzeta and NMII-A compete to bind directly to Lgl1 via the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α, and aPKCζ in polarized migrating cells (Dahan, 2013).
A model is proposed for the role of Lgl1-NMIIA and Lgl1-Par6α-aPKCζ in establishing front-rear polarization in migrating cells. In migrating polarized cells Lgl1 resides at the cell’s leading edge in a complex with Par6α-aPKCζ, and it is this complex which defines the leading edge of the cell. In the lamellipodium Lgl1 binds to NMII-A but not to aPKCζ, inhibiting NMII-A filament assembly. These events allow the cell to polymerize F-actin to move the cell forward. According to this model Lgl1 is absent from the rear part of the cell, allowing NMII-A to assemble into filaments to enable cell retraction (Dahan, 2013).
Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. This study reports that mice with embryonic neural stem-cell-specific deletion of Llgl1 Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. This study shows that LLGL1 directly binds to and promotes internalization of N-cadherin (see Drosophila Cadherin-N), and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1 (see Drosophila aPKC), restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells (Jossin, 2017).
Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. This study generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, the effects were examined of Lgl1 loss in the OB. First, the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB were determined. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, the development of the OB was investigated. Interestingly, reduced thickness of the mitral cell layer and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) was observed in the glomerular layer of the olfactory bulb. The results demonstrate that Lgl1 is required for the development of the olfactory bulb and the deletion of Lgl1 results in impaired olfaction in mice (Li, 2016).
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