Drosophila Tumor Suppressor Gene Research Articles

PATH-65. MOLECULAR SIGNATURE OF FAT1 RELATED MOLECULES IN GLIOMAS IN THE CONTEXT OF THE WHO 2016 CLASSIFICATION

Abstract Glioblastoma (GBM, WHO grade-IV) being the most malignant and aggressive form of glioma remains a major clinical challenge, with an overall 5-year survival rate of only 9.8%. Till recently, glioma diagnosis and grading were solely dependent on the phenotypic and histological features. However, with the advancement in the understanding of the molecular biology of glioma several molecules have been identified. The importance of these molecular/genotypic features of the tumor became evident by the inclusion of these molecular features by World Health Organization (WHO) in 2016 in glioma sub-grouping. Our lab is focused on studying the role of FAT1 gene (human ortholog of Drosophila tumor suppressor gene, fat) in glioma biology and aggressiveness. We observed FAT1 gene to have an oncogenic role in glioma where it has been found to upregulate migration/invasion, inflammatory microenvironment of the tumors, HIF1α expression/activity in the tumor-cells under severe hypoxia and in regulating EMT/stemness properties of GBM-cells under hypoxia. Here, we have characterized the molecular relationship between FAT1 related molecules and known- molecular markers of glioma with the hope of identifying glioma subgroup with a molecular signature of clinical significance by (i) analyzing the expression correlation of FAT1 and FAT1 regulated pro-inflammatroy molecules like COX2, IL1b and IL6 with the known- molecular markers of glioma like p53, IDH1, MGMT, EGFR, TERT in low-grade (grade-II) and high-grade (grade-III/IV) gliomas (n=50) by real-time PCR, sequencing, immunohistochemistry and in-silico analysis of TCGA-GBM-data (ii) Analyzed the regulatory role of FAT1 on the above known markers by siRNA mediated knockdown of FAT1 in in-vitro cell-culture system and (iii) further analyzed the identified molecular signature for their correlation with the patients prognosis/survival in the follow up patients. We observed a novel molecular signature with significant correlation with patients’ clinical outcome. Therapeutic targetting of FAT1 may benefit patients with high FAT1 expressing tumors.

Tumorigenic Properties of Drosophila Epithelial Cells Mutant for lethal giant larvae.

Mutations in Drosophila tumor suppressor genes (TSGs) lead to the formation of invasive tumors in the brain and imaginal discs. Here we studied the tumorigenic properties of imaginal discs mutant for the TSG gene lethal giant larvae (lgl). lgl mutant cells display the characteristic features of mammalian tumor cells: they can proliferate indefinitely, induce additional tracheogenesis (an insect counterpart of vasculogenesis) and invade neighboring tissues. Lgl mutant tissues exhibit high apoptotic levels, which lead to the activation of the Jun-N-Terminal Kinase (JNK) pathway. We propose 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 tumor cells capacity to invade wild-type tissue. Noteworthy, lgl RNAi-mediated down-regulation does not produce similar transformations in the central nervous system (CNS), thereby indicating a fundamental difference between the cells of developing imaginal discs and those of differentiated organs. We discuss these results in the light of the "single big-hit origin" of some human pediatric or developmental cancers. Down-regulation of lgl in imaginal discs is sufficient to enhance tracheogenesis and to promote invasion and colonization of other larval structures including the CNS. Developmental Dynamics 245:834-843, 2016. © 2016 Wiley Periodicals, Inc.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Abstract 4102: FAT1: A novel regulator of cancer and inflammation.

Abstract FAT1, a member of cadherin superfamily is a large trans-membrane protein with 34 cadherin repeats. FAT1 was originally identified as a tumor suppressor gene in Drosophila but its role in human cancers is just beginning to unfold. Considering its tumor suppressive role in Drosophila, few studies have reported similar function of FAT1 in human cancers but on the contrary several studies have also demonstrated oncogenic role of FAT1. In this study we identified oncogenic role of FAT1 in human glioma where knockdown of FAT1 was found to inhibit migration and invasion of glial cells. Further investigation into molecular mechanism of FAT1 function revealed direct link to AP-1 mediated transcription via Programmed Cell Death 4 (PDCD4). Knockdown of FAT1 was found to inhibit AP-1 mediated transcription and expression of downstream target genes. AP-1 is a known mediator of cancer and inflammation and accordingly we found decreased expression of several pro-inflammatory markers like COX-2, cytokines like IL-6, IL-1β and chemokines like IL-8, MCP-1 and MIP-2 after FAT1 knockdown. Interestingly, the expression of anti-inflammatory cytokine IL-10 was found to be increased after FAT1 knockdown. Moreover FAT1 expression was also found to positively correalate with COX-2 and IL-6 expression in primary GBM tumors. Thus, in this study FAT1 was found to act as an oncogene as well as to orchestrate the inflammatory milieu of the glioma cells by regulating AP-1 mediated transcription via PDCD4. Citation Format: Bhawana Dikshit, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1: A novel regulator of cancer and inflammation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2013-4102

Abstract 4942: Deletion of the asymmetry regulator Lgl1 contributes to tumor stemness and invasiveness.

Abstract In the developing and adult brain asymmetric cell division (ACD) maintains a balance between self-renewing and differentiated progeny of neural stem and progenitor cells. The WD protein lethal giant larvae 1 (Lgl1) regulates the distribution of polarity proteins in dividing cells and thereby establishes an axis of polarity, which is crucial for proper asymmetric divisions to occur (Baek et al. Mutat Res. 1999; 436; 131-6). Lgl, along with other asymmetry regulators, have been identified as a putative tumor suppressor gene in Drosophila. There remains debate as to whether disrupting ACD by itself is sufficient to cause tumor initiation by mammalian stem and progenitor cells. We have previously shown that expression of multiple asymmetry regulators is disrupted in human glioma (Sugiarto et al. Cancer Cell; 2011; 20; 320-340). Here we address our hypothesis that ACD defects are crucial for tumorigenesis, by delineating the role of Lgl1 in tumor initiation and progression. Using mouse neural stem cells (NSCs) isolated from genetically modified Lgl1lox/lox mice (Klezovitch et al. Genes Dev. 2004; 18; 559-71) we investigated the contribution of Lgl1 to various hallmarks of cancer in vitro and in vivo. We demonstrate that loss of Lgl1 in mouse NSC and oligodendrocyte precursor cells disrupts ACD and increases the expression of early-stage precursor cell markers. Deletion of Lgl1 does not increase proliferation in vitro and deletion of Lgl1 alone does not transform cells transplanted into immunocompromised mice. However properties associated with a high-grade tumor, including invasiveness and stem cell marker expression, are increased when Lgl1 is deleted in the context of oncogenic EgfrVIII expression. Increase in tumor invasiveness has previously been demonstrated to be associated with an increase in stem cell marker expression (Molina et al. Neoplasia. 2010; 12; 453-63). Our data suggest that loss of asymmetry in tumor precursor cells blocks differentiation and contributes to tumor progression by increasing stemness and hence invasiveness. Citation Format: Robin G. Lerner, Loan Nguyen, Claudia Petritsch. Deletion of the asymmetry regulator Lgl1 contributes to tumor stemness and invasiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2013-4942

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

Abstract A38: Overexpression of FAT1 in human GBM (glioblastoma multiforme) and high-grade glioma cell lines

Abstract FAT1, a human homologue of Drosophila tumor suppressor gene fat, is located on chromosome 4q35 in humans. In Drosophila, fat gene is known to be acting as an apical regulator of the tumor suppressor signaling, Salvador-Warts-Hippo (SWH) pathway. In mammalian cells, FAT1 is known to have a role in cell migration. The role of FAT1 in cancer is not clear and not much literature is available. In this study, we analyzed the expression of FAT1 in GBM (glioblastoma multiforme, grade IV glioma) samples at both mRNA and protein levels by means of q-PCR and immunohistochemistry. Herein, we demonstrate that 51.4% (18/35) of GBM samples had high expression of FAT1 mRNA, and 53.33% (8/15) of GBM samples had high FAT1 protein expression. We also checked the expression of FAT1 in glioma cell lines by q-PCR. Of the six glioma cell lines analyzed, four were GBM cell lines (U87MG, A172, U373MG and T98G) and two were astrocytoma grade 3 cell lines (GOS3 and SW1088). All GBM cell lines had high FAT1 expression while grade 3 cell lines had no or very low FAT1 expression. Since U87MG had maximum FAT1 expression we chose this cell line to study the effects of FAT1 knockdown by siRNA. FAT1 knockdown resulted in altered morphology and reduced migration of U87MG cells as compared to control siRNA treated cells. Since FAT1 is known to be an apical regulator of hippo pathway in Drosophila, we checked the expression of hippo pathway molecules by q-PCR and found that Mst2 (mammalian homolog of Drosophila hippo) expression was reduced up to 65% while other molecules like LATS and YAP were not altered at mRNA level. Considering the fact that regulation of SWH pathway primarily occurs at posttranslational level and depends upon phosphorylation status of the molecules, we are in process of analyzing the expression of these molecules at protein level and their phosphorylation status using phosphospecific antibodies, which would provide better understanding of the role of FAT1 in influencing SWH pathway. Since FAT1 is overexpressed in 50% of GBM samples studied and also found to have high expression in high-grade glioma cell lines as compared to low-grade glioma cell lines, it might act as an oncogene and may be involved in progression from low-grade to high-grade glioma. Thus, we propose FAT1 as a novel molecular target for therapeutic intervention in glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A38.

Hippo signaling in Drosophila: Recent advances and insights

The Hippo signaling pathway emerged from studies of Drosophila tumor suppressor genes, and is now appreciated as a major growth control pathway in vertebrates as well as arthropods. As a recently discovered pathway, key components of the pathway are continually being identified, and new insights into how the pathway is regulated and deployed are arising at a rapid pace. Over the past year and a half, significant advances have been made in our understanding of upstream regulatory inputs into Hippo signaling, key negative regulators of Hippo pathway activity have been identified, and important roles for the pathway in regeneration have been described. This review describes these and other advances, focusing on recent progress in our understanding of Hippo signaling that has come from continued studies in Drosophila.

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion.

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ~2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5' upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.

Sds22/PP1 links epithelial integrity and tumor suppression via regulation of myosin II and JNK signaling

Loss of epithelial integrity often correlates with the progression of malignant tumors. Sds22, a regulatory subunit of protein phosphatase 1 (PP1), has recently been linked to regulation of epithelial polarity in Drosophila. However, its role in tumorigenesis remains obscure. In this study, using Drosophila imaginal tissue as an in vivo model system, we show that sds22 is a new potential tumor suppressor gene in Drosophila. Without sds22, cells lose epithelial architecture, and become invasive and tumorigenic when combined with Ras overexpression; conversely, sds22 overexpression can largely suppress tumorigenic growth of Ras(V12)scrib(-/-) mutant cells. Mechanistically, we show that sds22 prevents cell invasion and metastasis by inhibiting myosin II and Jun N-terminal kinase (JNK) activity downstream of PP1. Loss of this inhibition causes cells to lose epithelial organization and promotes cell invasion. Finally, human Sds22 is focally deleted and downregulated in multiple carcinomas, and this downregulation correlates with tumor progression, suggesting that sds22 inactivation may contribute to tumorigenesis and metastatic potential in human cancers via a similar mechanism.

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell–cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell–cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell–cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell–cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell–cell junctions and that these associations may regulate trafficking and processing of nectins.

Frequent epigenetic inactivation of KIBRA, an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network, is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in < 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 {t(12;21) (p13;q22)} chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.

Activation of a Novel Palmitoyltransferase through a t(6;14) Translocation In T/Myeloid Biphenotypic Leukemia and Subsets of Acute Myeloid Leukemia.

AbstractAbstract 1663The current World Health Organization (WHO) classification of hematologic malignancies includes a number of entities defined by specific genetic abnormalities. Most known abnormalities result from chromosomal translocations and mutations involving genes encoding transcription factors or tyrosine kinases. We identified the activation of a novel palmitoyltransferase, ZDHHC14, through a chromosomal translocation, t(6;14)(q25;q32). This translocation was initially documented by three independent groups in two children and two adult patients with T/myeloid biphenotypic leukemia, a poorly differentiated form of acute leukemia with a single population of leukemic blasts co-expressing antigens of both T-lymphoid and myeloid lineages. Recently, we detected the same translocation in two additional patients with T/myeloid biphenotypic leukemia. Molecular characterization of the translocation breakpoints revealed the juxtaposition of a regulatory region downstream of BCL11B (at chromosome region 14q32) upstream of an uncharacterized gene, ZDHHC14 (at chromosome region 6q25), leading to transcriptional activation of the ZDHHC14. Notably, this regulatory region from 14q32 has been found to be responsible for transcriptional activation of a homeobox gene, TLX3, as a result of a cryptic translocation t(5;14)(q35;q32) in T-cell acute lymphoblastic leukemia. Our subsequent survey of ZDHHC14 protein levels in blasts from patients with various subtypes of acute myeloid leukemia (AML) showed that the protein was not detectable in AML patients with t(8;21), t(15;17), or inv(16), but was detected in AML patients with FLT3 mutations or with complex karyotypes, indicating a possible association of increased expression of this protein with unfavorable prognostic features of the disease. We also demonstrated that overexpression of ZDHHC14 could inhibit TPA-induced cellular differentiation of K562 cells. Interestingly, the Drosophila homolog of ZDHHC14 has been found to be a palmitoyltransferase regulating Fat signaling. Fat is a Drosophila tumor suppressor gene, encoding a large cadherin that regulates growth and a form of tissue organization. While the function of ZDHHC14 and the role of protein palmitoylation in mammalian hematopoiesis remain unknown, recent studies in neuronal development have demonstrated that palmitoylation regulates diverse aspects of protein trafficking. Our findings suggest that palmitoyltransferase plays a previously unappreciated role in hematopoiesis and that increased expression of ZDHHC14 may be associated with a primitive status of leukocytes. Further characterization of ZDHHC14 may broaden our knowledge of leukemogenesis, contribute to risk stratification of acute leukemia, and provide a novel therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Retrograde BMP Signaling Controls Synaptic Growth at the NMJ by Regulating Trio Expression in Motor Neurons

Retrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ.

Role of Scrib and Dlg in anterior-posterior patterning of the follicular epithelium during Drosophila oogenesis

BackgroundProper patterning of the follicle cell epithelium over the egg chamber is essential for the Drosophila egg development. Differentiation of the epithelium into several distinct cell types along the anterior-posterior axis requires coordinated activities of multiple signaling pathways. Previously, we reported that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the posterior follicle cell (PFC) fate induction at mid-oogenesis. Here we explore the role of another two tumor suppressor genes, scribble (scrib) and discs large (dlg), in the epithelial patterning.ResultsWe found that removal of scrib or dlg function from the follicle cells at posterior terminal of the egg chamber causes a complete loss of the PFC fate. Aberrant specification and differentiation of the PFCs in the mosaic clones can be ascribed to defects in coordinated activation of the EGFR, JAK and Notch signaling pathways in the multilayered cells. Meanwhile, the clonal analysis revealed that loss-of-function mutations in scrib/dlg at the anterior domains result in a partially penetrant phenotype of defective induction of the stretched and centripetal cell fate, whereas specification of the border cell fate can still occur in the most anterior region of the mutant clones. Further, we showed that scrib genetically interacts with dlg in regulating posterior patterning of the epithelium.ConclusionIn this study we provide evidence that scrib and dlg function differentially in anterior and posterior patterning of the follicular epithelium at oogenesis. Further genetic analysis indicates that scrib and dlg act in a common pathway to regulate PFC fate induction. This study may open another window for elucidating role of scrib/dlg in controlling epithelial polarity and cell proliferation during development.

Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

BackgroundTumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.Principal FindingsWe have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.ConclusionsThese findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

The Vacuolar Proton Pump, V-ATPase, Is Required for Notch Signaling and Endosomal Trafficking in Drosophila

We have identified Rabconnectin-3alpha and beta (Rbcn-3A and B) as two regulators of Notch signaling in Drosophila. We found that, in addition to disrupting Notch signaling, mutations in Rbcn-3A and B cause defects in endocytic trafficking, where Notch and other membrane proteins accumulate in late endosomal compartments. We show that Notch is transported to the surface of mutant cells and that signaling is disrupted after the S2 cleavage. Interestingly, the yeast homolog of Rbcn-3A, Rav1, regulates the V-ATPase proton pump responsible for acidifying intracellular organelles. We found that, similarly, Rbcn-3A and B appear to regulate V-ATPase function. Moreover, we identified mutants in VhaAC39, a V-ATPase subunit, and showed that they phenocopy Rbcn-3A and Rbcn-3B mutants. Our results demonstrate that Rbcn-3 affects Notch signaling and trafficking through regulating V-ATPase function, which implies that the acidification of an intracellular compartment in the receiving cells is crucial for signaling.

Discs large regulates somatic cyst cell survival and expansion in Drosophila testis

Gonad development requires a coordinated soma-germline interaction that ensures renewal and differentiation of germline and somatic stem cells to ultimately produce mature gametes. The Drosophila tumour suppressor gene discs large (dlg) encodes a septate junction protein functioning during epithelial polarization, asymmetric neuroblast division, and formation of neuromuscular junctions. Here, we report the role of dlg in testis development and its critical function in somatic cyst cells (SCCs). In these cells dlg is primarily required for their survival and expansion, and contributes to spermatocyte cyst differentiation. Cell death primarily occurred in SCCs at the end of spermatogonial amplification at a time when Dlg becomes restricted in wild-type (wt) testes to the distal somatic cells capping the growing spermatocyte cysts. RNAi depletion of dlg transcripts in early SCCs fully prevented testis development, whereas depletion in late SCCs resulted in a breakdown of spermatocyte cyst structure and germ cell individualization. Specific dlg expression in SCCs resulted in developmental rescue of dlg mutant testes, whereas its expression in germ cells exerted no such effect. dlg overexpression in wt testes led to spermatocyte cyst expansion at the expense of spermatogonial cysts. Our data demonstrate that dlg is essentially required in SCCs for their survival, expansion, and differentiation, and for the encapsulation of the germline cells.

Phosphorylation of the Tumor Suppressor Fat Is Regulated by Its Ligand Dachsous and the Kinase Discs Overgrown

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.