Decreased expression of lethal giant larvae causes ovarian follicle cell outgrowth in the Drosophila Scutoid mutant.
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ABSTRACT:
SUBMITTER: Tseng CY
PROVIDER: S-EPMC5737974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it 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. Our 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.
| S-EPMC5829493 | biostudies-literature
Project description:Schistosomiasis is a neglected tropical disease of humans, and it is considered to be the second most devastating parasitic disease after malaria. Eggs produced by normally developed female worms are important in the transmission of the parasite, and they responsible for the pathogenesis of schistosomiasis. The tumor suppressor gene lethal giant larvae (lgl) has an essential function in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. In our earlier study, downregulation of the lgl gene induced a significant reduction in the egg hatching rate of Schistosoma japonicum (Sj) eggs. In this study, the Sjlgl gene was used as a vaccine candidate against schistosomiasis, and vaccination achieved and maintained a stable reduction of the egg hatching rate, which is consistent with previous studies, in addition to reducing the worm burden and liver egg burden in some trials.
| S-EPMC5120214 | biostudies-literature
Project description:Cancer is a complex phenomenon involving multiple cellular pathways and processes in various combinations. As a genetic system, Drosophila has contributed much to our understanding of processes that initiate tumorigenesis. However, these are still disparate threads of information and a comprehensive understanding of how these various events and processes come together to bring about tumorous transformation is still wanting. The present study is an attempt to create a comprehensive picture of the dynamic process of epithelial tumour development in imaginal discs using lgl mutants as a model of neoplastic transformation. Experimental Procedure: The experiments were carried out in isogenized w-; lgl4. FRT40A/CyO. Actin –GFP (E Gateff and Schneiderman 1974). Fairly homogenous cultures of flies were added to population cages 2-3 days post eclosion for egg collection. Eggs were collected on yeasted agar plates which were kept in the cages for 1 to 1.5 hours. Plates with eggs were kept at 25 degrees for 21 hours, after which they were gently washed under running water to remove early hatched larvae. The plates were then yeasted again and kept at 25 degrees for another 24 hours to let all the eggs hatch. The food with the 24 hour old larvae was then transferred to large food-grade plastic boxes that had a single cotton plugged hole on the lid to allow for proper ventilation. These boxes also had standard fly food and were well yeasted. The larvae were allowed to grow in these boxes at 25 degrees until the time they were harvested. No other synchronization process was done on these cultures. The boxes containing synchronized larval cultures were washed in gently running water and the water along with the washed off larvae were collected in a beaker. The larvae were allowed to sink to the bottom and the water was decanted off. This was repeated many times till the decanted water was clear of food and other debris. Finally, a 3M solution of NaCl was poured into the beaker. This causes the larvae to float to the surface along with the large pieces of food left over from the water washes. Water was then gently added to the beaker till the concentration of the salt solution was just enough to allow only the larvae to float on the surface while the food pieces sunk to the bottom. At this point, the solution, along with the floating larvae was decanted off through a sieve to collect the larvae. These were then transferred to a glass petri-plate and washed with distilled water to remove all traces of salt solution. The larvae were finally floated in PBS for GFP selection under fluorescence microscope. The selected larvae were transferred to glass cavity blocks rinsed in RNAzap RNAse Inhibitor solution containing RNA-ase free PBS and dissected. For isolating imaginal discs, larvae were cut from the middle with the help of forceps and scissors, and then flipped inside out with needles so that the inner body wall is completely exposed with the imaginal discs attached. Fat bodies and other internal organs were carefully removed with the help of the forceps and needles. The imaginal discs/tumors were dissected out from these flipped larvae, care being taken to remove all traces of fat bodies or other debris from the discs and immediately transferred to RNAse free Microfuge tubes. The tubes were centrifuged briefly to collect the imaginal discs/tumors at the bottom while the PBS was pipetted off. The discs were then resuspended in 150-300 uL of RLT Buffer (Qiagen) in which 1.5-3 uL of Beta-mercaptoethanol was added. The tubes were then vortexed to lyse the cells. This suspension was stored at -40 degrees until RNA preparation. Total RNA was prepared from the lysed wing discs/tumors suspended in the guanidine thiocynate containing Lysis Buffer (RLT) supplied with the RNeasy Mini Kit and 14.3M beta-Mercaptoethanol using the ethanol precipitation/column filtration protocol recommended for the RNeasy Mini Kit by the Manufacturer. The purified total RNA was quantified and its quality checked by spectrophotometry and Agarose Gel electrophoresis respectively. Total RNA from 30-50 pairs of imaginal discs/tumors were used to make a single sample depending on the tumor stage with RNA yields typically ranging from 10 ug to 15 ug. Purified total RNA passing the quality controls were immediately used for labeling and hybridization reactions. The total RNA was processed and hybridized to Affymetrix GeneChip Drosophila Whole Genome 2.0 arrays using the manufacturer’s protocol, and the arrays were scanned immediately after hybridization.
2021-02-18 | E-MTAB-10059 | biostudies-arrayexpress
Project description:Lethal Giant Larvae (LGL) is a cytosolic cell polarity scaffold whose loss dominantly enhances neuromuscular junction (NMJ) synaptic overgrowth caused by loss of the Fragile X Mental Retardation Protein (FMRP). However, direct roles for LGL in NMJ morphological and functional development have not before been tested. Here, we use confocal imaging and two-electrode voltage-clamp electrophysiology at the Drosophila larval NMJ to define the synaptic requirements of LGL. We find that LGL is expressed both pre- and postsynaptically, where the scaffold localizes at the membrane on both sides of the synaptic interface. We show that LGL has a cell autonomous presynaptic role facilitating NMJ terminal branching and synaptic bouton formation. Moreover, loss of both pre- and postsynaptic LGL strongly decreases evoked neurotransmission strength, whereas the frequency and amplitude of spontaneous synaptic vesicle fusion events is increased. Cell-targeted RNAi and rescue reveals separable pre- and postsynaptic LGL roles mediating neurotransmission. We show that presynaptic LGL facilitates the assembly of active zone vesicle fusion sites, and that neuronally targeted rescue of LGL is sufficient to ameliorate increased synaptic vesicle cycling imaged with FM1-43 dye labeling. Postsynaptically, we show that loss of LGL results in a net increase in total glutamate receptor (GluR) expression, associated with the selective elevation of GluRIIB subunit-containing receptors. Taken together, these data indicate that the presynaptic LGL scaffold facilitates the assembly of active zone fusion sites to regulate synaptic vesicle cycling, and that the postsynaptic LGL scaffold modulates glutamate receptor composition and function.
| S-EPMC3666253 | biostudies-literature
Project description:Motile cilia perform crucial functions during embryonic development and throughout adult life. Development of organs containing motile cilia involves regulation of cilia formation (ciliogenesis) and formation of a luminal space (lumenogenesis) in which cilia generate fluid flows. Control of ciliogenesis and lumenogenesis is not yet fully understood, and it remains unclear whether these processes are coupled. In the zebrafish embryo, lethal giant larvae 2 (lgl2) is expressed prominently in ciliated organs. Lgl proteins are involved in establishing cell polarity and have been implicated in vesicle trafficking. Here, we identified a role for Lgl2 in development of ciliated epithelia in Kupffer's vesicle, which directs left-right asymmetry of the embryo; the otic vesicles, which give rise to the inner ear; and the pronephric ducts of the kidney. Using Kupffer's vesicle as a model ciliated organ, we found that depletion of Lgl2 disrupted lumen formation and reduced cilia number and length. Immunofluorescence and time-lapse imaging of Kupffer's vesicle morphogenesis in Lgl2-deficient embryos suggested cell adhesion defects and revealed loss of the adherens junction component E-cadherin at lateral membranes. Genetic interaction experiments indicate that Lgl2 interacts with Rab11a to regulate E-cadherin and mediate lumen formation that is uncoupled from cilia formation. These results uncover new roles and interactions for Lgl2 that are crucial for both lumenogenesis and ciliogenesis and indicate that these processes are genetically separable in zebrafish.
| S-EPMC3596994 | biostudies-literature
Project description:In Par complex-mediated cell polarity, phosphorylation by atypical protein kinase C (aPKC) is coupled to substrate cortical displacement. Polarized substrates often contain multiple phosphorylation sites, but the role of multisite phosphorylation in Par-mediated polarity remains unclear. Here, we have dissected the role of the three aPKC phosphorylation sites within the tumor suppressor Lethal giant larvae. Using a cultured Drosophila S2 cell cortical displacement assay, we observed that phosphorylation at any one site causes only partial displacement. Complete displacement requires that all three sites be modified. We undertook a kinetic analysis to determine if aPKC phosphorylates each site equivalently. As the sites are closely spaced, we observed not only differences in the rate of phosphorylation but also interaction between the sites. A complete description of the rates reveals a preferential order of phosphorylation. Our results provide new insights into how multiple phosphorylations and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.
| S-EPMC4372073 | biostudies-literature
Project description:Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.
| S-EPMC2976283 | biostudies-literature
Project description:Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.
| S-EPMC8688431 | biostudies-literature
Project description:The present study is a pioneering investigation that has been carefully designed to study proteomic effects of specific cancer-related mutation using the lgl mutant of D. melanogaster that has not been well characterised thus far. Recent findings concerning the D. melanogaster cancer models suggest that this model is well suited for study of cancer mechanisms and therapeutic interventions, especially in the brain and intestines (Mirzoyan et al., 2019). In addition, the integration of circadian biology into cancer research offers new insights into the implications of optimal timing for cancer treatment that could potentially reduce undesirable side effects and enhance prognosis (Ballesta et al., 2017). As a result, label free quantitative mass spectrometry was employed to investigate tissue-specific protein expression in head and intestinal tissues of D. melanogaster lgl mutants which was collected at 6-hours intervals throughout the 24-hour period. Furthermore, the efficacy of melatonin as an anticancer agent at the proteome level was also investigated. Together, the resulting information could potentially identify malfunctioning signalling pathways and circadian rhythm-dependent protein markers during tumorigenesis, as well as the mechanism of actions of melatonin on these processes, post-treatment
2023-05-10 | PXD033191 | Pride
Project description:BackgroundNeoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.ResultsWe observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.ConclusionsOur results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.
| S-EPMC2877678 | biostudies-literature