Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2.
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ABSTRACT:
SUBMITTER: Almagor L
PROVIDER: S-EPMC6561149 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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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.
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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.
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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.
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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.
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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: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.
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Project description:FUBP-interacting repressor (FIR) is a suppressor of transcription of the proto-oncogene MYC. FIR binds to the far upstream element (FUSE) of the MYC promoter. Competition of FIR with FUSE-binding protein 1 (FUBP1) is a key mechanism of MYC transcriptional regulation. To gain insights into the structural mechanisms regulating FIR DNA interaction, we determined the crystal structure of two FIR RRM domains (RRM1-2) with single-stranded FUSE DNA sequences. These structures revealed an ability of the RRM domain to recognize diverse FUSE regions through distinct intermolecular interactions and binding modes. Comparative structural analyses against available RRM-ssDNA/RNA complexes showed that the nucleotide configurations in FIR were similar to those in other RRMs that harbour a tyrosine at the conserved aromatic position in the RNP2 motif (Y-type RRM), but not those with a phenylalanine (F-type RRM). Site-directed mutagenesis experiments demonstrated that a single substitution, Y115F, altered the binding affinities of oligonucleotides to FIR RRM, suggesting an important role of this conserved aromatic residue in ssDNA/RNA interactions. Our study provides the structural basis for further mechanistic studies on this important protein-DNA interaction.
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Project description:Atypical protein kinase C (aPKC) isoforms ? and ? interact with polarity complex protein Par3 and are evolutionarily conserved regulators of cell polarity. Prkcz encodes aPKC-? and PKM-?, a truncated, neuron-specific alternative transcript, and Prkcl encodes aPKC-?. Here we show that, in embryonic hippocampal neurons, two aPKC isoforms, aPKC-? and PKM-?, are expressed. The localization of these isoforms is spatially distinct in a polarized neuron. aPKC-?, as well as Par3, localizes at the presumptive axon, whereas PKM-? and Par3 are distributed at non-axon-forming neurites. PKM-? competes with aPKC-? for binding to Par3 and disrupts the aPKC-?-Par3 complex. Silencing of PKM-? or overexpression of aPKC-? in hippocampal neurons alters neuronal polarity, resulting in neurons with supernumerary axons. In contrast, the overexpression of PKM-? prevents axon specification. Our studies suggest a molecular model wherein mutually antagonistic intermolecular competition between aPKC isoforms directs the establishment of neuronal polarity.
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