The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis.

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.     

The multiple-wing-hairs gene encodes a novel GBD-FH3 domain-containing protein that functions both prior to and after wing hair initiation

The frizzled signaling/signal transduction pathway controls planar cell polarity (PCP) in both vertebrates and invertebrates. Epistasis experiments argue that in the Drosophila epidermis multiple wing hairs (mwh) acts as a downstream component of the pathway. The PCP proteins accumulate asymmetrically in pupal wing cells where they are thought to form distinct protein complexes. One is located on the distal side of wing cells and a second on the proximal side. This asymmetric protein accumulation is thought to lead to the activation of the cytoskeleton on the distal side, which in turn leads to each cell forming a single distally pointing hair. We identified mwh as CG13913, which encodes a novel G protein binding domain–formin homology 3 (GBD–FH3) domain protein. The Mwh protein accumulated on the proximal side of wing cells prior to hair formation. Unlike planar polarity proteins such as Frizzled or Inturned, Mwh also accumulated in growing hairs. This suggested that mwh had two temporally separate functions in wing development. Evidence for these two functions also came from temperature-shift experiments with a temperature-sensitive allele. Overexpression of Mwh inhibited hair initiation, thus Mwh acts as a negative regulator of the cytoskeleton. Our data argued early proximal Mwh accumulation restricts hair initiation to the distal side of wing cells and the later hair accumulation of Mwh prevents the formation of ectopic secondary hairs. This later function appears to be a feedback mechanism that limits cytoskeleton activation to ensure a single hair is formed.     

The Drosophila javelin gene encodes a novel actin-associated protein required for actin assembly in the bristle

The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development.     

fear of intimacy encodes a novel transmembrane protein required for gonad morphogenesis in Drosophila

Gonad formation requires specific interactions between germ cells and specialized somatic cells, along with the elaborate morphogenetic movements of these cells to create an ovary or testis. We have identified mutations in the fear of intimacy (foi) gene that cause defects in the formation of the embryonic gonad in DROSOPHILA: foi is of particular interest because it affects gonad formation without affecting gonad cell identity, and is therefore specifically required for the morphogenesis of this organ. foi is also required for tracheal branch fusion during tracheal development. E-cadherin/shotgun is similarly required for both gonad coalescence and tracheal branch fusion, suggesting that E-cadherin and FOI cooperate to mediate these processes. foi encodes a member of a novel family of transmembrane proteins that includes the closely related human protein LIV1. Our findings that FOI is a cell-surface protein required in the mesoderm for gonad morphogenesis shed light on the function of this new family of proteins and on the molecular mechanisms of organogenesis.     

The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast.

Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.     

The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells

ADP-ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP-ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site-directed mutagenesis demonstrates that the ADP-ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP-ribosylating toxin critical for the pathogenesis of Salmonella infections.     

The three rows gene of Drosophila melanogaster encodes a novel protein that is required for chromosome disjunction during mitosis.

Zygotic expression of the three rows (thr) gene of Drosophila melanogaster is required for normal cell proliferation during embryogenesis. Mitotic defects in thr mutant embryos begin during mitosis 15, and all subsequent divisions are disrupted. Chromosome disjunction and consequently cytokinesis fail during these defective mitoses, although the initial mitotic processes (chromosome condensation, spindle assembly, metaphase plate formation, and cyclin degradation) are not affected. Despite the failure of chromosome disjunction and cytokinesis, later mitotic events (chromosome decondensation) and subsequent cell cycle progression continue. The thr gene has been isolated and shown to encode a 1209 amino acid protein that shares no extended sequence similarity with known proteins. thr mRNA is present as maternal mRNA that degrades at the time of cellularization. At this and all subsequent times during embryogenesis, zygotic expression correlates with mitotic proliferation. These observations, together with the observation that the zygotic phenotype of thr mutant embryos is influenced by the maternal genotype, suggest that the embryonic phenotype results from exhaustion of the maternal thr contribution and does not reflect a developmentally restricted requirement for thr function. Our results indicate that the novel thr product is required specifically for chromosome disjunction during all mitoses.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.     

The Drosophila gastrulation gene concertina encodes a G alpha-like protein

Gastrulation is a complex process requiring the coordination of cell shape changes and cell movements. In Drosophila, gastrulation begins immediately upon cellularization of the blastoderm stage embryo with the formation of the ventral furrow and posterior midgut. Cells that form both of these invaginations change their shape via apical constriction. Embryos from mothers homozygous for mutations in the concertina (cta) gene begin furrow formation by forming a zone of tightly apposed cells, constrict some cells, and then fail to constrict enough cells to form an organized groove. The cta gene has been cloned, and sequence analysis suggests that it encodes an alpha subunit of a G protein. G proteins have a role in cell-cell communication as mediators of signals between membrane-bound receptors and intracellular effectors. The phenotype of embryos from homozygous cta mothers suggests that the cta gene plays a role in a signal transduction pathway used during gastrulation.     

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis.     

The Drosophila spn-D gene encodes a RAD51C-like protein that is required exclusively during meiosis

In Drosophila, mutations in double-strand DNA break (DSB) repair enzymes, such as spn-B, activate a meiotic checkpoint leading to dorsal-ventral patterning defects in the egg and an abnormal appearance of the oocyte nucleus. Mutations in spn-D cause an array of ovarian phenotypes similar to spn-B. We have cloned the spn-D locus and found that it encodes a protein of 271 amino acids that shows significant homology to the human RAD51C protein. In mammals the spn-B and spn-D homologs, XRCC3 and RAD51C, play a role in genomic stability in somatic cells. To test for a similar role for spn-B and spn-D in double-strand DNA repair in mitotic cells, we analyzed the sensitivity of single and double mutants to DSBs induced by exposure to X rays and MMS. We found that neither singly mutant nor doubly mutant animals were significantly sensitized to MMS or X rays. These results suggest that spn-B and spn-D act in meiotic recombination but not in repair of DSBs in somatic cells. As there is no apparent ortholog of the meiosis-specific DMC1 gene in the Drosophila genome, and given their meiosis-specific requirement, we suggest that spn-B and spn-D may have a function comparable to DMC1.     

The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.     

A gene located at 71F in Drosophila melanogaster encodes a novel zinc-finger protein that interacts with protein kinase CK2

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are two hormones produced and secreted by the heart to control blood pressure, body fluid homeostasis and electrolyte balance. Each peptide binds to a common family of 3 receptors (GC-A, GC-B and C-receptor) with varying degrees of affinity. The proANP gene disrupted mouse model provides an excellent opportunity to examine the regulation and expression of BNP in the absence of ANP. A new radioimmunoassay (RIA) was developed in order to measure mouse BNP peptide levels in the plasma, atrium and ventricle of the mouse. A detection limit of 3–6 pg/tube was achieved by this assay. Results show that plasma and ventricular level of BNP were unchanged among the three genotypes of mice. However, a significant decrease in the BNP level was noted in the atrium. The homozygous mutant (ANP–/–) had undetectable levels of BNP in the atrium, while the heterozygous (ANP+/–) and wild-type (ANP+/+) mice had 430 and 910 pg/mg in the atrium, respectively. Northern Blot analysis shows the ANP–/– mice has a 40% reduction of BNP mRNA level in the atrium and a 5-fold increase in the ventricle as compared with that of the ANP+/+ mouse. Our data suggest that there is a compensatory response of BNP expression to proANP gene disruption. Despite the changes in the atrial and ventricular tissue mRNA and peptide levels, the plasma BNP level remains unaltered in the ANP–/– mice. We conclude that the inability of BNP to completely compensate for the lack of ANP eventually leads to chronic hypertension in the proANP gene disrupted mice.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.