Bristle Morphology Research Articles

Twinfilin, a molecular mailman for actin monomers.

Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains. It forms a 1:1 complex with ADP-actin-monomers, inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments. The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites. Twinfilin also interacts with PtdIns(4,5)P(2), which inhibits its actin-monomer-sequestering activity in vitro. Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S. cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster. These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin. Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their 'inactive' ADP-form, to the sites of rapid filament assembly. This is mediated through direct interactions between twinfilin and capping protein. Therefore, twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer–binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.

Genetic analysis of functional domains within the Drosophila LARK RNA-binding protein.

LARK is an essential Drosophila RNA-binding protein of the RNA recognition motif (RRM) class that functions during embryonic development and for the circadian regulation of adult eclosion. LARK protein contains three consensus RNA-binding domains: two RRM domains and a retroviral-type zinc finger (RTZF). To show that these three structural domains are required for function, we performed a site-directed mutagenesis of the protein. The analysis of various mutations, in vivo, indicates that the RRM domains and the RTZF are required for wild-type LARK functions. RRM1 and RRM2 are essential for viability, although interestingly either domain can suffice for this function. Remarkably, mutation of either RRM2 or the RTZF results in the same spectrum of phenotypes: mutants exhibit reduced viability, abnormal wing and mechanosensory bristle morphology, female sterility, and flightlessness. The severity of these phenotypes is similar in single mutants and double RRM2; RTZF mutants, indicating a lack of additivity for the mutations and suggesting that RRM2 and the RTZF act together, in vivo, to determine LARK function. Finally, we show that mutations in RRM1, RRM2, or the RTZF do not affect the circadian regulation of eclosion, and we discuss possible interpretations of these results. This genetic analysis demonstrates that each of the LARK structural domains functions in vivo and indicates a pleiotropic requirement for both the LARK RRM2 and RTZF domains.

Mallomonas nieringii sp. nov., a new species of Synurophyceae from a suite of ponds on Cape Cod, Massachusetts, U.S.A.

A new species of Mallomonas, M. nieringii sp. nov., is described from a small, poorly buffered, acidic locality on Cape Cod, Massachusetts, U.S.A. Cells are covered with uniquely sculptured scales, each scale of which may possess a single bristle. Cell, scale and bristle morphology all indicate that this new species should be placed within the series Punctiferae of the section Punctiferae, which now has four recognized species and one variety. Scales of the apical ring are highly asymmetric in design, each with a forward projecting triangular shaped spine. Collectively, the triangular shaped extensions of the apical ring of scales surround a single emergent flagellum. Body scales resemble those of Mallomonas punctifera and M. transsylvanica in shape and general morphology, but differ in lacking distinct secondary ribbing on the shield. Mallomonas nieringii was found in six waterbodies all situated within a small geographic area on the outer tip of the Cape Cod peninsula.

Cloning and characterization of a new theta-class glutathione-S-transferase (GST) gene, gst-3, from Drosophila melanogaster.

We report here on the cloning and characterization of a new theta-class glutathione-S-transferase (GST) gene, gst-3, from Drosophila melanogaster. Its sequence is distinct from previously characterized Drosophila GST genes, and Southern blotting shows no other closely related genes in the genome. In-situ hybridization localizes the gene to chromosome 2 (55D), near gst-2 (53F), and clearly separate from the gst-D cluster at 87B. The gene is intronless and appears to possess conventional 5' TATA, Cap and 3' polyadenylation signals. A single transcript, approximately 1kb in size, appears to be expressed at high levels in all developmental stages examined. When this gene is overexpressed using various upstream GAL4 driver systems, no striking phenotypes are observed; however, we detect bristle morphology defects in some progeny. The gst-3 gene does not appear to be essential, based upon our observation that mutant flies homozygous for an EP element insertion 5' to the TATA box produce little or no detectable gst-3 mRNA; these flies are viable and fertile at 25 and 29 degrees C. Nevertheless, the gst-3 gene appears to be evolutionarily conserved in other Drosophila species, suggesting that it may be functionally important.

Mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development

Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3epsilon, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene.

Skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release.

The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.

Changes in the F-actin cytoskeleton during neurosensory bristle development in Drosophila: the role of singed and forked proteins.

Drosophila neurosensory bristle development provides an excellent model system to study the role of the actin-based cytoskeleton in polarized cell growth. We used confocal fluorescence microscopy of isolated thoracic tissue to characterize changes in F-actin that occurred during macrochaete development in wild type flies and mutants that have aberrant bristle morphology. At the earliest stages in wild type bristle development, cortical patches of F-actin were present, but no bundles were observed. Actin bundles began to form at 31% of pupal development and became more prominent as development progressed. The F-actin patches gradually disappeared and were no longer present by 38% of pupal development. The distribution of F-actin in singed3 mutant macrochaetae was indistinguishable from wild type bristles until 35% of development when the actin bundles began to splay and appear ribbon-like. In forked36a bristles, the mutant phenotype was evident at earlier stages of development than the singed3 mutant. Wild type tissue stained with antibodies against the forked protein demonstrated that the forked protein colocalized with F-actin structures found in early and late stage developing macrochaetae. Antibodies against the singed protein showed it appeared to localize with F-actin structures only at later stages in development. These data suggested that the forked gene product was required for the initiation of fiber bundle formation and the singed gene product was required for the maintenance of fiber bundle morphology during bristle development. Similar analyses of singed3/forked36a double mutants provided additional genetic evidence that the forked gene product was required before the singed gene product. Further, the analyses suggested that at least one additional crosslinking protein was present in these bundles.

Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila.

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.

Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies.

Transmembrane ligands can be internalized across cell boundaries into receptor-expressing cells. In the developing Drosophila eye imaginal disc, the bride of sevenless transmembrane protein (boss) is expressed on the surface of R8 cells. After internalization into neighboring R7 cells, the boss protein accumulates in multivesicular bodies. In a search for genes that affect this cell-type-specific pattern of boss endocytosis, we found that mutations in the hook gene inhibit the accumulation of boss in multivesicular bodies of R7 cells. In addition, hook flies exhibit pleiotropic phenotypes including abnormal bristle morphology and eye degeneration. The wild-type-pattern of boss endocytosis was restored in hook mutants by a genomic rescue fragment containing the hook gene or by a hook cDNA expressed in R7 cells under control of a sevenless (sev) enhancer. The hook gene encodes a novel cytoplasmic protein of 679 amino acids with a central coiled-coil domain of some 200 amino acids. Truncated, epitope-tagged hook proteins coimmunoprecipitated the full-length protein, indicating dimerization mediated by the coiled-coil domain. The hook protein localizes to vesicular structures that are part of the endocytic compartment. The requirement of the hook protein in R7 cells for the accumulation of boss protein in multivesicular bodies, and the localization of the hook protein to endocytic vesicles indicate that the hook gene encodes a novel component of the endocytic compartment that plays an important role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies.

Evidence for intrinsic differences in the formation of chromatin domains in Drosophila melanogaster.

The results of an investigation into intrinsic differences in the formation of two different heterochromatic domains are presented. The study utilized two different position effect variegation mutants in Drosophila melanogaster for investigating the process of compacting different stretches of DNA into heterochromatin. Each stretch of DNA encodes for a gene that affects different aspects of bristle morphology. The expression of each gene is prevented when it is compacted into heterochromatin thus the genes serve as effective reporter systems to monitor the spread of heterochromatin. Both variegating mutants are scored in the same cell such that environmental and genetic background differences are unambiguously eliminated. Any differences observed in the repression of the two genes must therefore be the result of intrinsic differences in the heterochromatic compaction process for the two stretches of DNA. Studies of the effects different enhancers of variegation have upon the compaction of the two genes indicate each compaction event occurs independently of the other, and that different components are involved in the two processes. These results are discussed with regard to spreading heterochromatin and the role this process may play in regulating gene expression.

The l(3)S12 locus of Drosophila melanogaster: heterochromatic position effects and stage-specific misexpression of the gene in P element transposons.

l(3)S12 is a vital locus whose function is required in embryos, early larvae, late pupae and oogenesis. We have identified a cold-sensitive allele, l(3)S12(3), and characterized conditional misexpression of the gene associated with this mutation as well as with several euchromatic insertions of l(3)S12+ transposons. Surviving cold-sensitive mutants as well as underexpression variants generated by P element transformation display a phenotypic syndrome that can include delayed development, abnormal bristle morphology, and female sterility. Using these phenotypes, defects in putative "early" and "late" l(3)S12 expression can be identified. The sensitivity of certain l(3)S12+ insertions to site-specific euchromatic position effect appears to be due to separation of the gene from an endogenous enhancer element during cloning. This enhancerless construct can be used to identify and perhaps to select "permissive" euchromatic sites, presumably adjacent to enhancer elements, which in some cases permit elevated production not only of the l(3)S12 message, but also of a P element-l(3)S12 fusion transcript. Certain of these permissive sites appear to control stage-specific expression, and we propose that this system may be used to identify, clone, and characterize such loci. Heterochromatic position effect on this locus has been demonstrated. Available evidence suggests that the l(3)S12 gene may be involved in protein synthesis, perhaps encoding a ribosomal protein.

Temperature effect on the morphological S character related to DSV hereditary reovirus in Drosophila simulans

The effects of temperature changes during the development on the bristle morphology of Drosophila simulans carrying the hereditary S virus (DSV) have been studied. Taking into account that S phenotype (anomalous bristles) is an indication of the existence of DSV in flies, the main findings were: (i) Extreme temperatures (17° and 28°C) produce an increase of wild phenotype flies. The effects are greater at 28°C (almost 100% of normal flies) than at 17°C. (ii) The main effect of DSV on bristle development seems to occur at the beginning of its formation. (iii) A significant effect on bristle elongation may also be detected. These facts support the causative effect of DSV on bristle development. The results are discussed bearing in mind the known actions of reoviruses on the cytoskeleton and protein synthesis.

Mallomonas alphaphora (Chrysophyceae), a new species from Western Australia

A new species ofMallomonas, M. alphaphora (Chrysophyceae), was found in freshwater ponds in the Perth region, Western Australia. It is distinguished from other species ofMallomonas by its very distinctive scale and bristle morphology and is placed in a new section,Alphaphorae, of the genusMallomonas.

Colonization of North America by the European Species, Drosophila subobscura and D. ambigua

Two European species, Drosophila subobscura and D. ambigua, have been found in the Pacific Northwest. Drosophila subobscura occurs from central California, N to southwestern British Columbia. Drosophila ambigua has been found only in the southwestern mainland of British Columbia. These species can be distinguished from the endemic obscura-subgroup species, D. pseudoobscura, D. persimilis and C. miranda, by sex comb, vaginal plate and wing bristle morphology, by allozymic differences and by differences in the polytene chromosomes. INTRODUCTION Students of the North American Drosophila obscura species group are not accustomed to viewing members of the group as cosmopolitan entities. The usual methods of species identification (Anderson et al., 1977; Crumpacker, 1973) simply provide means of distinguishing among the known endemic species. Thus difficulties arise when distinct species previously unknown from this continent begin appearing in collections. Two such species appeared in collections from the Pacific coastal region, from northern California to southern British Columbia. After ruling out the known North American representatives, analysis of metaphase chromosome configurations and hybridization results with D. pseudoobscura, D. miranda and D. persimilis suggested that the new residents are the widespread European species, D. subobscura and D. ambigua. Subsequent crosses to known European strains of these species verified the identifications. This colonization is not the first for Drosophila subobscura. In February 1978, it was found in Puerto Montt, Chile, and has since spread in South America to include a region from 29 ?-53 S lat (Budnik and Brncic, 1982). It has also spread eastward into Argentina (Prevosti et al., 1983). Its Old World range includes Europe from southern Scandinavia S to northern Africa and the Canary Islands, eastward to Iran, and westward to the Azores Islands (Prevosti, 1978). Drosophila ambigua occurs from southern Finland, S to the Iberian Peninsula and E to Greece (Lakovaara and Saura, 1982). The purpose of this paper is to report the apparently successful colonization by these two species in the Pacific Northwest. Morphological, allozymic and chromosomal means of distinguishing them from the indigenous Drosophila obscura subgroup species are presented, along with the presently known North American ranges of each. MATERIALS AND METHODS Collections were made at buckets of fermenting bananas. Females were immediately isolated individually into vials of standard medium of corn meal, yeast and sugars. In most collections, the species were identified by analyzing polytene chromosomes from third instar larvae from each female. The three indigenous species were identified by third and XL chromosomes (Anderson et al., 1977). The polytenes of Drosophila subobscura and D. ambigua bear little resemblance to one another or to those of any member of

Expression and amplification of the genes for ribosomal RNA in bobbed mutants ofDrosophila melanogaster

SummaryWe have employed stocks bearing clonally derivedXchromosomes to investigate several features of the bobbed mutant syndrome, and the amplification of rDNA genes inD. melanogaster. We report that posterior macroscutellar bristle length correlates well with the rDNA content (i.e. dose ofivs–, or uninterrupted genes) in clonedXderivative strains.X/Omales andX/Xfemales with statistically indistinguishable rDNA contents have virtually identical bristle lengths. This indicates that (with respect to this phenotypic character) the rDNAs in these two genotypes are expressed equally, without apparent sexual dimorphism or dosage compensation. However, the severity of bobbed phenotype in terms of bristle morphology, turgite etching, and delayed eclosion is greater in theXbb/XNO−female than in theXbb/Omale genotype for the alleles examined. We estimate the minimum dose of functioning rRNA genes required for viability at 26 δC to be 70 genes per diploid genome. We have examined the capacity of severalXchromosomes which bear bobbed mutant alleles to compensate inX/Omales, and find that disproportionate replication of these rDNAs does not take place. In contrast, at least one of the non-compensating bobbed alleles does appear to undergo rDNA magnification.

Developmental analysis of fs(1)1867, an egg resorption mutation of Drosophila melanogaster.

The X-linked 1867+ gene seems to be a pleiotropic one. Mutation in this gene causes delay in development and abnormal bristle morphology. These phenotypes are expressed autonomously in genetic mosaics. There is no focus for the delay. The female sterility could be localized to the ovary (based on ovary transplantations). It seems that the 1867+ gene is expressed in the follicular cells at one of the last steps of oogenesis. This is suggested by the results of mosaic analysis based on mitotic recombination. Possible drawbacks of the mitotic recombination type of analyses are also discussed.

STUDIES ON THE EXPRESSION OF VARIOUS SINGED ALLELES IN DROSOPHILA MELANOGASTER

RECENT work on a set of alleles that pleiotropically affect both setal morphology and oogenesis has provided a complex of data that have not been systematically analyzed. These alleles constitute the sex-linked mutation singed. In 1951, IVES and NOYES demonstrated 0.04 percent crossing over between sn3 and sdnk. Genes such as these which are physically close together, similar in function, and yet separable by crossing over are called pseudoalleles. Position pseudoallelism (LEWIS 1942) was demonstrated among the singed mutants by HEXTER (1955) who showed that sns +/+ sn’ flies displayed a singed phenotype while sn3 sn‘/ ++ flies were wild type in appearance. Subsequently he demonstrated three loci in the sn complex which he preliminarily characterized ( HEXTER 1957). Twenty-eight singed mutations have been reported since MOHR’S first record ( 1922), but at present only 11 are available, and these were used in the following study. These stocks and progeny of the possible combinations were examined for bristle morphology, fertility, and the anatomy of the female reproductive tract. The classes of phenotypes were analyzed; this phenotypic array of gene effect was then used together with crossover data to provide a model for the loci involved.