Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Cell autonomous polarization by the planar cell polarity signaling pathway.

Planar Cell Polarity (PCP) signaling polarizes epithelial cells in a plane orthogonal to their apical-basal axis. A core PCP signaling module both generates molecular asymmetry within cells and coordinates the direction of polarization between neighboring cells. Two subcomplexes of core proteins segregate to opposite sides of the cell, defining a polarity axis. Homodimers of the atypical cadherin Flamingo are thought to be the scaffold upon which these subcomplexes assemble and are required for intercellular polarity signaling. The central role for Flamingo homodimers in scaffolding and intercellular communication suggests that cells in which intercellular signaling via Flamingo is disabled should fail to polarize. We show that cells lacking Flamingo, or bearing a truncated Flamingo that cannot homodimerize do in fact polarize. Cell polarization requires both positive and negative feedback, and in a multicellular tissue, feedback might involve both intracellular and intercellular pathways. We identify positive and negative feedback pathways that operate cell autonomously to drive polarization.

Protein phosphatase 1 regulates core PCP signaling.

PCP signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of Protein Phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one Serine/Threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Asymmetric distribution of prickle-like 2 reveals an early underlying polarization of vestibular sensory epithelia in the inner ear.

Vestibular hair cells have a distinct planar cell polarity (PCP) manifest in the morphology of their stereocilia bundles and the asymmetric localization of their kinocilia. In the utricle and saccule the hair cells are arranged in an orderly array about an abrupt line of reversal that separates fields of cells with opposite polarity. We report that the putative PCP protein Prickle-like 2 (Pk2) is distributed in crescents on the medial sides of vestibular epithelial cells before the morphological polarization of hair cells. Despite the presence of a line of polarity reversal, crescent position is not altered between hair cells of opposite polarity. Frizzled 6 (Fz6), a second PCP protein, is distributed opposite Pk2 along the lateral side of vestibular support cells. Similar to Pk2, the subcellular localization of Fz6 does not differ between cells located on opposite sides of the line of reversal. In addition, in Looptail/Van Gogh-like2 mutant mice Pk2 is distributed asymmetrically at embryonic day 14.5 (E14.5), but this localization is not coordinated between adjacent cells, and the crescents subsequently are lost by E18.5. Together, these results support the idea that a conserved PCP complex acts before stereocilia bundle development to provide an underlying polarity to all cells in the vestibular epithelia and that cells on either side of the line of reversal are programmed to direct the kinocilium in opposite directions with respect to the polarity axis defined by PCP protein distribution.

Thirty-one flavors of Drosophila rab proteins.

Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, >75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. We have begun a systematic genetic study of the 33 Rab genes in Drosophila. Most of the fly proteins are clearly related to specific vertebrate proteins. We report here the creation of a set of transgenic fly lines that allow spatially and temporally regulated expression of Drosophila Rab proteins. We generated fluorescent protein-tagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. We describe Drosophila Rab expression patterns during embryogenesis, the subcellular localization of some Rab proteins, and comparisons of the localization of wild-type, dominant-negative, and constitutively active forms of selected Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful tool kit for investigating Rab functions in vivo.

Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development.

Drosophila Costal1 mutations are alleles of protein kinase A that modulate hedgehog signaling.

Hedgehog (Hh) signaling is crucial for the development of many tissues, and altered Hh signal transduction can result in cancer. The Drosophila Costal1 (Cos1) and costal2 (cos2) genes have been implicated in Hh signaling. cos2 encodes a kinesin-related molecule, one component of a cytoplasmic complex of Hh signal transducers. Mutations in Cos1 enhance loss-of-function cos2 mutations, but the molecular nature of Cos1 has been unknown. We found that previously identified alleles of Cos1 actually map to two separate loci. Four alleles of Cos1 appear to be dominant-negative mutations of a catalytic subunit of protein kinase A (pka-C1) and the fifth allele, Cos1(A1), is a gain-of-function allele of the PKA regulatory subunit pka-RII. PKA-RII protein levels are higher in Cos1(A1) mutants than in wild type. Overexpression of wild-type pka-RII phenocopies Cos1 mutants. PKA activity is aberrant in Cos1(A1) mutants. PKA-RII is uniformly overproduced in the wing imaginal disc in Cos1(A1) mutants, but only certain cells respond by activating the transcription factor Ci and Hh target gene transcription. This work shows that overexpression of a wild-type regulatory subunit of PKA is sufficient to activate Hh target gene transcription.

Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.

Regulation of ribosome biogenesis by nucleostemin 3 promotes local and systemic growth in Drosophila.

Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.

Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis.

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell's apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

Rab35 controls actin bundling by recruiting fascin as an effector protein.

Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.

Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling.

BACKGROUND: Mutation of Wnt signal antagonists Apc or Axin activates beta-catenin signaling in many cancers including the majority of human colorectal adenocarcinomas. The phenotype of apc or axin mutation in the fruit fly Drosophila melanogaster is strikingly similar to that caused by mutation in the segment-polarity gene, naked cuticle (nkd). Nkd inhibits Wnt signaling by binding to the Dishevelled (Dsh/Dvl) family of scaffold proteins that link Wnt receptor activation to beta-catenin accumulation and TCF-dependent transcription, but human NKD genes have yet to be directly implicated in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We identify for the first time mutations in NKD1--one of two human nkd homologs--in a subset of DNA mismatch repair-deficient colorectal tumors that are not known to harbor mutations in other Wnt-pathway genes. The mutant Nkd1 proteins are defective at inhibiting Wnt signaling; in addition, the mutant Nkd1 proteins stabilize beta-catenin and promote cell proliferation, in part due to a reduced ability of each mutant Nkd1 protein to bind and destabilize Dvl proteins. CONCLUSIONS/SIGNIFICANCE: Our data raise the hypothesis that specific NKD1 mutations promote Wnt-dependent tumorigenesis in a subset of DNA mismatch-repair-deficient colorectal adenocarcinomas and possibly other Wnt-signal driven human cancers.

A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size.

Growth and body size are regulated by the CNS, integrating the genetic developmental program with assessments of an animal's current energy state and environmental conditions. CNS decisions are transmitted to all cells of the animal by insulin/insulin-like signals. The molecular biology of the CNS growth control system has remained, for the most part, elusive. Here we identify NS3, a Drosophila nucleostemin family GTPase, as a powerful regulator of body size. ns3 mutants reach <60% of normal size and have fewer and smaller cells, but exhibit normal body proportions. NS3 does not act cell-autonomously, but instead acts at a distance to control growth. Rescue experiments were performed by expressing wild-type ns3 in many different cells of ns3 mutants. Restoring NS3 to only 106 serotonergic neurons rescued global growth defects. These neurons are closely apposed with those of insulin-producing neurons, suggesting possible communication between the two neuronal systems. In the brains of ns3 mutants, excess serotonin and insulin accumulate, while peripheral insulin pathway activation is low. Peripheral insulin pathway activation rescues the growth defects of ns3 mutants. The findings suggest that NS3 acts in serotonergic neurons to regulate insulin signaling and thus exert global growth control.

Mes2, a MADF-containing transcription factor essential for Drosophila development.

The development of the Drosophila mesoderm is initiated by the basic helix-loop-helix transcription factor twist. We identified a gene encoding a putative transcription factor, mes2, in a screen for essential mesoderm-expressed genes that function downstream of twist. Mes2 protein belongs to a family of 48 Drosophila proteins containing MADF domains. MADF domains exist in worms, flies, and fish. Mes2 is a nuclear protein first produced in trunk and head mesoderm during late gastrulation. At later embryonic stages, mes2 is expressed in glia of the central and peripheral nervous systems, and in tissues derived from the head mesoderm. We have identified a null mutation of mes2 that leads to developmental arrest in first instar larvae. Increased production of Mes2 in multiple embryonic and larval tissues almost always causes lethality. The ubiquitous or epidermal misexpression of mes2 in the embryo causes a dramatic loss of epidermal integrity resulting in the failure of dorsal closure. Our data show that the precise regulation of mes2 expression is critical for normal development in Drosophila and implicate Mes2 in the regulation of essential target genes.

A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis.

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused.

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. We have analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. Our results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh.

Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction.

Hedgehog (Hh) signaling is critical for many developmental events and must be restrained to prevent cancer. A transmembrane protein, Smoothened (Smo), is necessary to transcriptionally activate Hh target genes. Smo activity is blocked by the Hh transmembrane receptor Patched (Ptc). The reception of a Hh signal overcomes Ptc inhibition of Smo, activating transcription of target genes. Using Drosophila salivary gland cells in vivo and in vitro as a new assay for Hh signal transduction, we investigated the regulation of Hh-triggered Smo stabilization and relocalization. Hh causes Smo to move from internal membranes to the cell surface. Relocalization is protein synthesis-independent and occurs within 30 min of Hh treatment. Ptc and the kinesin-related protein Costal2 (Cos2) cause internalization of Smo, a process that is dependent on both actin and microtubules. Disruption of endocytosis by dominant negative dynamin or Rab5 prevents Smo internalization. Fly versions of Smo mutants associated with human tumors are constitutively present at the cell surface. Forced localization of Smo at the plasma membrane activates Hh target gene transcription. Conversely, trapping of activated Smo mutants in the ER prevents Hh target gene activation. Control of Smo localization appears to be a crucial step in Hh signaling in Drosophila.