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1.  CRB3A Controls the Morphology and Cohesion of Cancer Cells through Ehm2/p114RhoGEF-Dependent Signaling 
Molecular and Cellular Biology  2015;35(19):3423-3435.
The transmembrane protein CRB3A controls epithelial cell polarization. Elucidating the molecular mechanisms of CRB3A function is essential as this protein prevents the epithelial-to-mesenchymal transition (EMT), which contributes to tumor progression. To investigate the functional impact of altered CRB3A expression in cancer cells, we expressed CRB3A in HeLa cells, which are devoid of endogenous CRB3A. While control HeLa cells display a patchy F-actin distribution, CRB3A-expressing cells form a circumferential actomyosin belt. This reorganization of the cytoskeleton is accompanied by a transition from an ameboid cell shape to an epithelial-cell-like morphology. In addition, CRB3A increases the cohesion of HeLa cells. To perform these functions, CRB3A recruits p114RhoGEF and its activator Ehm2 to the cell periphery using both functional motifs of its cytoplasmic tail and increases RhoA activation levels. ROCK1 and ROCK2 (ROCK1/2), which are critical effectors of RhoA, are also essential to modulate the cytoskeleton and cell shape downstream of CRB3A. Overall, our study highlights novel roles for CRB3A and deciphers the signaling pathway conferring to CRB3A the ability to fulfill these functions. Thereby, our data will facilitate further investigation of CRB3A functions and increase our understanding of the cellular defects associated with the loss of CRB3A expression in cancer cells.
PMCID: PMC4561736  PMID: 26217016
2.  Rac1 controls epithelial tube length through the apical secretion and polarity pathways 
Biology Open  2015;5(1):49-54.
The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.
Summary: Epithelial tube size regulation sustains organ physiology. Rac1 limits tube elongation in the fly trachea through restriction of apical membrane growth, and by supporting luminal secretion of chitin modifying enzymes.
PMCID: PMC4728308  PMID: 26700724
Epithelial tube morphogenesis; Epithelial polarity; Drosophila trachea; Rac1; Crumbs; Coracle; Septate junction
3.  Mouse Crumbs3 sustains epithelial tissue morphogenesis in vivo 
Scientific Reports  2015;5:17699.
The human apical protein CRB3 (Crb3 in mouse) organizes epithelial cell polarity. Loss of CRB3 expression increases the tumorogenic potential of cultured epithelial cells and favors metastasis formation in nude mice. These data emphasize the need of in vivo models to study CRB3 functions. Here, we report the phenotypic analysis of a novel Crb3 knockout mouse model. Crb3-deficient newborn mice show improper clearance of airways, suffer from respiratory distress and display perinatal lethality. Crb3 is also essential to maintain apical membrane identity in kidney epithelial cells. Numerous kidney cysts accompany these polarity defects. Impaired differentiation of the apical membrane is also observed in a subset of cells of the intestinal epithelium. This results in improper remodeling of adhesive contacts in the developing intestinal epithelium, thereby leading to villus fusion. We also noted a strong increase in cytoplasmic β-catenin levels in intestinal epithelial cells. β-catenin is a mediator of the Wnt signaling pathway, which is overactivated in the majority of colon cancers. In addition to clarifying the physiologic roles of Crb3, our study highlights that further functional analysis of this protein is likely to provide insights into the etiology of diverse pathologies, including respiratory distress syndrome, polycystic kidney disease and cancer.
PMCID: PMC4668553  PMID: 26631503
4.  A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity 
The Journal of Cell Biology  2014;204(4):487-495.
During epithelial cell polarization, aPKC phosphorylates Yurt to prevent its premature apical localization, while at the same time Yurt binds to and restrains aPKC function.
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.
PMCID: PMC3926957  PMID: 24515345
5.  Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death 
The Journal of Cell Biology  2012;198(6):991-998.
Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity.
Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.
PMCID: PMC3444775  PMID: 22965909
6.  Characterization of Fragile X Mental Retardation Protein Recruitment and Dynamics in Drosophila Stress Granules 
PLoS ONE  2013;8(2):e55342.
The RNA-binding protein Fragile X Mental Retardation (FMRP) is an evolutionarily conserved protein that is particularly abundant in the brain due to its high expression in neurons. FMRP deficiency causes fragile X mental retardation syndrome. In neurons, FMRP controls the translation of target mRNAs in part by promoting dynamic transport in and out neuronal RNA granules. We and others have previously shown that upon stress, mammalian FMRP dissociates from translating polysomes to localize into neuronal-like granules termed stress granules (SG). This localization of FMRP in SG is conserved in Drosophila. Whether FMRP plays a key role in SG formation, how FMRP is recruited into SG, and whether its association with SG is dynamic are currently unknown. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMR1 (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple model, we assessed the role of dFMRP in SG formation and defined the determinants required for its recruitment in SG as well as its dynamics in SG. We show that dFMRP is dispensable for SG formation in vitro and ex vivo. FRAP experiments showed that dFMRP shuttles in and out SG. The shuttling activity of dFMRP is mediated by a protein-protein interaction domain located at the N-terminus of the protein. This domain is, however, dispensable for the localization of dFMRP in SG. This localization of dFMRP in SG requires the KH and RGG motifs which are known to mediate RNA binding, as well as the C-terminal glutamine/asparagine rich domain. Our studies thus suggest that the mechanisms controlling the recruitment of FMRP into SG and those that promote its shuttling between granules and the cytosol are uncoupled. To our knowledge, this is the first demonstration of the regulated shuttling activity of a SG component between RNA granules and the cytosol.
PMCID: PMC3567066  PMID: 23408971
7.  Emerging Role for Epithelial Polarity Proteins of the Crumbs Family as Potential Tumor Suppressors 
Defects in apical-basal polarity regulation are associated with tissue overgrowth and tumorogenesis, yet the molecular mechanisms linking epithelial polarity regulators to hyperplasia or neoplasia remain elusive. In addition, exploration of the expression and function of the full complement of proteins required for the polarized architecture of epithelial cells in the context of cancer is awaited. This paper provides an overview of recent studies performed on Drosophila and vertebrates showing that apical polarity proteins of the Crumbs family act to repress tissue growth and epithelial to mesenchymal transition. Thus, these proteins emerge as potential tumor suppressors. Interestingly, analysis of the molecular function of Crumbs proteins reveals a function for these polarity regulators in junctional complexes stability and control of signaling pathways regulating proliferation and apoptosis. Thereby, these studies provide a molecular basis explaining how regulation of epithelial polarity is coupled to tumorogenesis.
PMCID: PMC3168773  PMID: 21912482
8.  Epithelial polarity proteins regulate Drosophila tracheal tube size independently of the luminal matrix pathway 
Current biology : CB  2010;20(1):55.
Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube-size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM) [1–4]. ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ) [5, 6]. Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube-size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins [7, 8], cause an expansion of tracheal tubes, but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes a SJ-associated polarity protein [8, 9]. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.
PMCID: PMC2821987  PMID: 20022244
Tracheal morphogenesis; Tubulogenesis; epithelial polarity; Crumbs; Yurt; Coracle Scribble; Septate junction; Vermiform
9.  The FERM Protein Yurt Is a Negative Regulatory Component of the Crumbs Complex that Controls Epithelial Polarity and Apical Membrane Size 
Developmental cell  2006;11(3):363-374.
The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.
PMCID: PMC2834949  PMID: 16950127
10.  Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells 
The Journal of Cell Biology  2005;169(4):635-646.
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.
PMCID: PMC2171699  PMID: 15897260

Results 1-10 (10)