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L Cooley

Publications and source records attributed to L Cooley.

At least 37 records · Page 2Linked to original sources
PubMed1997

Genetic analysis of the actin cytoskeleton in the Drosophila ovary.

The Drosophila ovary provides a favorable model system in which to study cellular morphogenesis. The development of a mature egg involves a syncytium of 16 germline cells and over 1000 somatically derived follicle cells. Intercellular transport, stable intercellular bridges, cell migrations, cell shape changes, and specific subcellular localization of many embryonic patterning determinants contribute to egg development and require a dynamic cytoskeleton. We discuss many of the recent genetic and cell biological studies that have led to insights into how the actin cytoskeleton is assembled and regulated during the morphogenesis of the Drosophila egg.

Actins↗
PubMed1997

Examination of the function of two kelch proteins generated by stop codon suppression.

The Drosophila kelch gene produces a single transcript with a UGA stop codon separating two open reading frames (ORF1 and ORF2). From the transcript, 76 kDa ORF1 and 160 kDa full-length (ORF1 + ORF2) proteins are made. The expression of these two proteins is regulated in a tissue-specific manner causing the ratio of full-length to ORF1 protein to vary in different tissues. The only detected defect for kelch mutants is female sterility, and kelch protein is localized to the ovarian ring canals. kelch mutant ring canals are disorganized and have partly occluded lumens, causing a failure to transport cytoplasm. ORF1 and full-length kelch proteins co-sediment with ring canals suggesting that both proteins are found in the ring canals. Transgenetic analysis reveals that ORF1 kelch protein is sufficient to rescue ring canal morphology and fertility. In addition, we have mutated the UGA stop codon to a UAA stop codon and to three sense codons that allow constitutive readthrough. Analysis of these mutants reveals that a full-length kelch protein can partially compensate for the loss of endogenous kelch, but the residue included at the stop codon is critical for function. Finally, these studies suggest that the mechanism of stop codon suppression of kelch is by tRNA suppression.

Alanine↗
PubMed1996

Stable intercellular bridges in development: the cytoskeleton lining the tunnel.

A wide variety of intercellular junctions that are involved with cell adhesion or signal transduction have been described in recent years. A widespread but less well-characterized type of intercellular junction is the stable intercellular bridge. Several organisms use stable intercellular bridges as cytoplasmic connections, probably to allow rapid transfer of information and organelles between cells. Here, the authors take a detailed look at the assembly of intercellular bridges called ring canals in the Drosophila germline and discuss how examination of mutants that disrupt Drosophila ovarian ring canal assembly indicates that these bridges are required for intercellular transport of cytoplasm.

Journal Article↗
PubMed1996

Single amino acid mutations in Drosophila fascin disrupt actin bundling function in vivo.

Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo; singedG409E mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo; singedS289N mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singedS289N sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.

Actins↗
PubMed1994

Cytoskeletal functions during Drosophila oogenesis.

Organismal morphogenesis is driven by a complex series of developmentally coordinated changes in cell shape, size, and number. These changes in cell morphology are in turn dependent on alterations in basic cytoarchitecture. Elucidating the mechanisms of development thus requires an understanding of the cytoskeletal elements that organize the cytoplasm of differentiating cells. Drosophila oogenesis has emerged as a versatile system for the study of cytoskeletal function during development. A series of highly coordinated changes in cytoskeletal organization are required to produce a mature Drosophila oocyte, and these cytoskeletal transformations are amenable to a variety of experimental approaches. Genetic, molecular, and cytological studies have shed light on the specific functions of the cytoskeleton during oogenesis. The results of these studies are reviewed here, and their mechanistic implications are considered.

Animals↗
PubMed1994

The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis.

Mutations in the Drosophila quail gene result in female sterility due to the disruption of cytoplasmic transport from the nurse cells into the oocyte late in oogenesis. Nurse cells from quail mutant egg chambers fail to assemble cytoplasmic actin filament bundles correctly. We have cloned the quail gene and found that it encodes a protein with homology to the vertebrate actin-regulating protein villin. Unlike vertebrate villin, which is restricted to specialized absorptive epithelial cells, the villin-like protein encoded by quail is germline specific in adult flies. Antibodies directed against the quail protein show a striking colocalization with filamentous actin in the nurse cells and the oocyte. Our results demonstrate that the villin-like product of quail is required for the formation of cytoplasmic actin filament bundles in nurse cells, possibly by regulating both the polymerization and organization of actin filaments as demonstrated for vertebrate villin in vitro.

Actins↗
PubMed1994

The specialized cytoskeleton of the Drosophila egg chamber.

The Drosophila egg chamber is emerging as a uniquely versatile system for studying cytoskeletal rearrangements during development. Initial determination of the oocyte fate and subsequent growth of the oocyte depend on a series of highly coordinated changes in cell architecture. Homologs or relatives of many known cytoskeletal proteins play key roles in these events.

Animals↗
PubMed1994

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.

Actins↗
PubMed1994

Profilin mutations disrupt multiple actin-dependent processes during Drosophila development.

The chickadee gene of Drosophila encodes profilin, a small actin binding protein. We present the first analysis of the effects of profilin deletion in a multicellular organism. Genomic deletions of the chickadee locus result in a late embryonic lethal phenotype indicating that profilin is essential in flies. In addition, viable alleles of chickadee with defects in oogenesis, spermatogenesis and bristle formation provide insight into profilin function in a variety of cell types. Defects in oogenesis include the previously described failure to assemble nurse cell actin filament bundles in addition to abnormal regulation of mitosis, binucleate cells and stalled cell migration. Malformed bristles are a result of aberrant actin assembly. Monoclonal antibodies against Drosophila profilin were generated to study profilin's cellular and subcellular localization.

Actins↗
PubMed1994

Morphogenesis of Drosophila ovarian ring canals.

We analyzed the structure of cytoplasmic bridges called ring canals in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. We raised antibodies against the carboxy-terminal tail of hts and found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the hts protein. kelch, which is localized to the ring canals hours after hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components.

Animals↗
PubMed1993

kelch encodes a component of intercellular bridges in Drosophila egg chambers.

Oocyte maturation in Drosophila is supported by a cluster of 15 germline-derived nurse cells whose cytoplasm is transported into the oocyte through intercellular bridges called ring canals. kelch was isolated as a female sterile mutation affecting cytoplasm transport. We have cloned the kelch gene and found that it encodes an unusual transcript containing two open reading frames (ORF1 and ORF2) separated by a single UGA stop codon. At least two protein products are made from the kelch mRNA: a short protein from ORF1 and a longer protein from both ORF1 and ORF2 as a result of partial suppression of the UGA codon. The kelch ORF1 product is conserved, and antibodies directed against it are localized specifically to ring canals. Our results suggest that kelch produces a component of ring canals that regulates the flow of cytoplasm between cells.

Amino Acid Sequence↗
PubMed1992

chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.

The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport. We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin. The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells.

Amino Acid Sequence↗
PubMed1990

Constructing deletions with defined endpoints in Drosophila.

Chromosomes bearing small deletions are valuable tools in Drosophila genetics. We have investigated a method for efficiently constructing precise chromosomal deficiencies. Two P transposable elements were positioned within a progenitor strain at the sites of the desired deletion endpoints. Deletions spanning the two transposons were recovered at high frequency when P element transposase was expressed in these flies, but only if the flanking P elements were in a cis rather than a trans configuration. Appropriate progenitor strains can now be constructed to delete virtually any chromosomal region by utilizing an extensive collection of lines containing single P element insertions throughout the Drosophila genome.

Animals↗