Gemin8 is required for the architecture and function of the survival motor neuron complex

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) in higher eukaryotes requires the functions of several cellular proteins and includes nuclear as well as cytoplasmic phases. In the cytoplasm, a macromolecular complex containing the survival motor neuron (SMN) protein, Gemin2-8 and Unrip mediates the ATP-dependent assembly of Sm proteins and snRNAs into snRNPs. To carry out snRNP assembly, the SMN complex binds directly to both Sm proteins and snRNAs; however, the contribution of the individual components of the SMN complex to its composition, interactions, and function is poorly characterized. Here, we have investigated the functional role of Gemin8 using novel monoclonal antibodies against components of the SMN complex and RNA interference experiments. We show that Gemin6, Gemin7, and Unrip form a stable cytoplasmic complex whose association with SMN requires Gemin8. Gemin8 binds directly to SMN and mediates its interaction with the Gemin6/Gemin7 heterodimer. Importantly, loss of Gemin6, Gemin7, and Unrip interaction with SMN as a result of Gemin8 knockdown affects snRNP assembly by impairing the SMN complex association with Sm proteins but not with snRNAs. These results reveal the essential role of Gemin8 for the proper structural organization of the SMN complex and the involvement of the heteromeric subunit containing Gemin6, Gemin7, Gemin8, and Unrip in the recruitment of Sm proteins to the snRNP assembly pathway.     

The Drosophila melanogaster importin alpha3 locus encodes an essential gene required for the development of both larval and adult tissues

The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression.     

Drosophila signal peptide peptidase is an essential protease for larval development

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.     

Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.     

drICE is an essential caspase required for apoptotic activity in Drosophila cells.

Caspases are involved in the execution of cell death in all multicellular organisms so far studied, including the nematode worm, fruit fly and vertebrates. While Caenorhabditis elegans has only a single identified caspase, CED-3, whose activity is absolutely required for all developmental programmed cell deaths, most mammalian cell types express multiple caspases with varying specificities. The fruit fly Drosophila melanogaster is genetically tractable, less complex than vertebrates and possesses two known caspases, DCP-1 and drICE. The fly may therefore provide a good model system for examining the hierarchy and relative roles of individual caspases in the execution of apoptosis. We have examined the role of drICE in in vitro apoptosis of the D.melanogaster cell line S2. We show that cytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper (rpr) expression or cycloheximide treatment contain a caspase activity with DEVD specificity which can cleave p35, lamin DmO, drICE and DCP-1 in vitro, and which can trigger chromatin condensation in isolated nuclei. Using antibodies specific to drICE, we show that immunodepletion of drICE from these lysates is sufficient to remove most measurable in vitro apoptotic activity, and that re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. We conclude that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis.     

Drosophila myosin V is required for larval development and spermatid individualization

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex.     

Talin is essential for integrin function in Drosophila

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.     

Embryonic expression of the divergent Drosophila beta3-tubulin isoform is required for larval behavior

We have sought to define the developmental and cellular roles played by differential expression of distinct beta-tubulins. Drosophila beta3-tubulin (beta3) is a structurally divergent isoform transiently expressed during midembryogenesis. Severe beta3 mutations cause larval lethality resulting from failed gut function and consequent starvation. However, mutant larvae also display behavioral abnormalities consistent with defective sensory perception. We identified embryonic beta3 expression in several previously undefined sites, including different types of sensory organs. We conclude that abnormalities in foraging behavior and photoresponsiveness exhibited by prelethal mutant larvae reflect defective beta3 function in the embryo during development of chordotonal and other mechanosensory organs and of Bolwig's organ and nerve. We show that microtubule organization in the cap cells of chordotonal organs is altered in mutant larvae. Thus transient zygotic beta3 expression has permanent consequences for the architecture of the cap cell microtubule cytoskeleton in the larval sensilla, even when beta3 is no longer present. Our data provide a link between the microtubule cytoskeleton in embryogenesis and the behavioral phenotype manifested as defective proprioreception at the larval stage.     

Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression,histone methylation and elongation factor recruitment

Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. We knocked down the catalytic subunit of P-TEFb, Cdk9, in Drosophila melanogaster using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation.     

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP- Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion- disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.     

Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation

SPD-2 is a C. elegans centriolar protein required for both centriole duplication and pericentriolar material (PCM) recruitment [1-4]. SPD-2 is conserved in Drosophila (DSpd-2) and is a component of the fly centriole [5-7]. The analysis of a P element-induced hypomorphic mutation has shown that DSpd-2 is primarily required for PCM recruitment at the sperm centriole but is dispensable for both centriole duplication and aster formation [5]. Here we show that null mutations carrying early stop codons in the DSpd-2 coding sequence suppress astral microtubule (MT) nucleation in both neuroblasts (NBs) and spermatocytes. These mutations also disrupt proper Miranda localization in dividing NBs, as previously observed in mutants lacking astral MTs [8-10]. Spermatocyte analysis revealed that DSpd-2 is enriched at both the centrioles and the PCM and is required for the maintenance of cohesion between the two centrioles but not for centriole duplication. We found that DSpd-2 localization at the centrosome requires the wild-type activity of Asl but is independent of the function of D-PLP, Cnn, gamma-tubulin, DGrip91, and D-TACC. Conversely, DSpd-2 mutants displayed normal centrosomal accumulations of Asl and D-PLP, strongly reduced amounts of Cnn, gamma-tubulin, and DGrip91, and diffuse localization of D-TACC. These results indicate that DSpd-2 functions in a very early step of the PCM recruitment pathway.     

MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis, but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis, and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis, and interactions with myocardium in the heart. We also found that MEKK3 is required for angiopoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild-type and Mekk3-deficient tumor-like embryonic stem cell lines and transplanted them subcutaneously into nude mice to assess their ability to grow and induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as control Mekk3 wild-type tumors and were also capable of inducing tumor angiogenesis. In addition, we found no differences in the production of VEGF in Mekk3-deficient tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation and survival and is required for endothelial cells to interact with the myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis. cardiovascular; mitogen-activated protein kinase; embryonic development     

NOP3 is an essential yeast protein which is required for pre-rRNA processing

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.     

The Drosophila embargoed gene is required for larval progression and encodes the functional homolog of schizosaccharomyces Crm1

The CRM1 (Exportin 1) protein is a receptor for leucine-rich nuclear export signal sequences. We have molecularly characterized the Drosophila melanogaster embargoed (emb) gene and find that it encodes a product with 49 and 71% sequence identity to the fission yeast Schizosaccharomyces pombe and human CRM1 proteins, respectively. We show that expression of the emb cDNA is sufficient to suppress the growth phenotype of both conditional-lethal and null S. pombe crm1(-) mutant strains, suggesting that emb encodes the functional homologue of the S. pombe Crm1 protein. Through mutagenesis screens we have recovered a series of recessive lethal emb mutations. There is a substantial maternal contribution of emb mRNA and animals hemizygous for our emb alleles can develop to second instar larvae but persist at this stage and consistently fail to undergo the molt to the third instar stage. We see a nuclear accumulation of endogenous actin in the intestinal epithelial cells of the emb mutant larvae, consistent with a role for the emb gene product in nuclear export of actin protein.     

Hsc70 is required for endocytosis and clathrin function in Drosophila

By screening for Drosophila mutants exhibiting aberrant bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, we have recovered a point mutation in Hsc70-4, the closest homologue to bovine clathrin uncoating ATPase. Although the mutant allele was lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrated that the Sevenless-mediated internalization of Boss was blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes was also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interacted genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibited diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs.     

Aldehyde dehydrogenase is essential for both adult and larval ethanol resistance in Drosophila melanogaster

The enzyme aldehyde dehydrogenase (ALDH) is essential for ethanol metabolism in mammals, converting the highly toxic intermediate acetaldehyde to acetate. The role of ALDH in Drosophila has been debated, with some authors arguing that, at least in larvae, acetaldehyde detoxification is carried out mainly by alcohol dehydrogenase (ADH), the enzyme responsible for converting ethanol to acetaldehyde. Here, we report the creation and characterization of four null mutants of Aldh, the putative structural locus for ALDH. Aldh null larvae and adults are poisoned by ethanol concentrations easily tolerated by wild-types; their ethanol sensitivity is in fact comparable to that of Adh nulls. The results refute the view that ALDH plays only a minor role in ethanol detoxification in larvae, and suggest that Aldh and Adh may be equally important players in the evolution of ethanol resistance in fruit-breeding Drosophila.     

The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival

Using a transposon insertion line of the Drosophila Genome Project we have cloned the black-pearl gene (blp), analyzed cDNA clones, generated various mutants, and characterized their phenotypes. The blp gene codes for a protein of 15.7 kDa calculated molecular weight that has been conserved from yeast to plants and mammals with high homology. A domain of these new proteins shows distant similarity to DnaJ domains indicating a functionally relevant interaction with other proteins. The P element insertion in line P1539 lies within the 5' untranslated leader of the black-pearl gene. Flies homozygous for this insertion are semi-lethal, escapers produce very few offspring and show melanotic inclusions in the hemocoel ('black pearls') similar to various melanotic 'tumor' mutants. Two small deletions confined to the blp gene and two EMS-induced mutations are homozygous lethal. These null mutants appear normal up to a prolonged first instar larval stage but fail to grow and die. Thus in Drosophila the blp gene is specifically required for larval growth. The evolutionary conservation in both unicellular and multicellular organisms suggests for the new protein family described here a fundamental role in cell growth.