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Biomedical subjects

N H Brown

Publications and source records attributed to N H Brown.

At least 19 recordsLinked to original sources
PubMed1998

kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin.

Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.

Amino Acid Sequence↗
PubMed1998

Modulation of integrin activity is vital for morphogenesis.

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the alpha subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (alphaPS2betaPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type alphaPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the alphaPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the alphaPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin alphaIIbbeta3.

Amino Acid Sequence↗
PubMed1998

Absence of PS integrins or laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos.

We have examined the role of integrins in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). To this end we have analyzed muscle attachments and neuromuscular junctions ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions (HAJs) to extracellular matrix, but not for their intracellular link to the cytoskeleton. The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. It is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component laminin A, while adhesion mediated by PS2 integrin does not. At neuromuscular junctions the formation of functional synapses occurs normally in embryos lacking PS integrins and/or laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly. We suggest that neuromuscular contact in part requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of laminin A.

Animals↗
PubMed1998

Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions.

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. In order to investigate whether PS2 integrin is required at these additional sites and/or has functions besides mediating adhesion, a comprehensive genetic analysis of inflated, the gene that encodes alphaPS2, was performed. We isolated 35 new inflated alleles, and obtained 10 alleles from our colleagues. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signalling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing.

Alleles↗
PubMed1998

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.

Alleles↗
PubMed1998

Guidelines and disease management.

The four NCCN Guidelines that follow represent the initial deliberations of an institutionally diverse, multidisciplinary panel comprised of internationally recognized experts. These experts have attempted to address the broad range of clinical decisions that oncologists face as they attempt to manage a patient with a particular tumor. The goal of the NCCN guidelines program is to publish comprehensive pathways that will serve as a foundation for all cancer care providers to develop superior disease management programs.

Disease Management↗
PubMed1997

Specificity of PS integrin function during embryogenesis resides in the alpha subunit extracellular domain.

We tested the ability of different integrin alpha subunits to substitute for each other during embryonic development. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers where they are required for normal development. Using the GAL4 system, we are able to rescue fully the embryonic lethality of an alphaPS2 null mutation with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, due to partial rescue of both muscle and midgut phenotypes. Similarly we are able to rescue the embryonic/first instar larval lethality of an alphaPS1 null mutation gene with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. We conclude that the two alpha subunits are not equivalent and have distinct functions which reside in the extracellular domains.

Amino Acid Sequence↗
PubMed1996

Intracellular signals direct integrin localization to sites of function in embryonic muscles.

In the Drosophila embryo, the alphaPS2betaPS integrin heterodimer is localized tightly at the termini of the multinucleate muscles where they attach to the alphaPS1betaPS-containing epidermal tendon cells. Here we examine the basis for alphaPS2betaPS integrin subcellular localization. We show that the betaPS cytoplasmic tail is sufficient to direct the localization of a heterologous transmembrane protein, CD2, to the muscle termini in vivo. This localization does not occur via an association with structures set up by the endogenous betaPS integrins, since it can occur even in the absence of the betaPS protein. Furthermore, the subcellular localization of the alphaPS2betaPS integrin is not dependent on any other interactions between the muscles and the tendon cells. In embryos that lack the segmental tendon cells, due to a mutation removing the related segment polarity genes engrailed and invected, alphaPS2betaPS is still localized to the muscle termini even though the ventral longitudinal muscles are not attached to the epidermis, but instead are attached end to end. Thus the alphaPS2betaPS integrin can be localized by an intracellular mechanism within the muscles. Our results challenge the view that the transmission of signals from the extracellular environment via integrins is required for the organization of the cytoskeleton and the resultant cellular polarity.

Amino Acid Sequence↗
PubMed1995

Mammalian CD2 is an effective heterologous marker of the cell surface in Drosophila.

Ectopic expression of neutral proteins, such as beta-galactosidase, in developing embryos has been an invaluable tool for studies of gene expression and embryonic development. However, expression of beta-galactosidase does not reveal the shape of the cells containing it. We have examined the suitability of rat CD2, a small transmembrane protein of the immunoglobulin superfamily, as a marker of cell morphology in Drosophila. We selected the regulatory sequences of the Drosophila mesoderm-specific gene twist to express CD2 and prepared a chimeric gene, twi-CD2. Embryos containing twi-CD2 faithfully express CD2 in the same pattern as Twist. Expression of CD2 on the surface of cells reveals the shape of cells when stained with existing monoclonal antibodies. We have also constructed a CD2 gene that can be used with the GAL4 system and show that CD2 can be expressed on the surface of epithelial cells and along the length of axons.

Animals↗
PubMed1995

Anterior-posterior subdivision and the diversification of the mesoderm in Drosophila.

We have used a novel cell marker, in which the twist promoter directs the synthesis of the cell surface protein CD2 (twi-CD2) to examine the development of the mesoderm in the Drosophila embryo after gastrulation and to locate the progenitor cell populations for different mesodermal derivatives. We find that the early mesoderm in each segment is divided into a more anterior region with relatively low levels of twist and twi-CD2 expression and a more posterior region where twist and twi-CD2 expression are high. This subdivision coincides with regional assignments of cells to form different progenitors: dorsal anterior cells invaginate to form an internal layer from which the visceral mesoderm is derived. Ventral anterior cells form progenitors of mesodermal glial cells. Dorsal posterior cells form heart. Ventral and dorsal posterior cells form somatic muscles. We conclude that the metamerically repeated anterior-posterior subdivision of the mesoderm is an essential element in laying out the pattern of mesodermal progenitor cells and in distinguishing between an internal cell layer which will give rise to the progenitors of visceral muscles and an external layer which will generate the somatic muscles and the heart.

Animals↗
PubMed1994

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10.

Morphogenesis in the yeast Saccharomyces cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Amino Acid Sequence↗
PubMed1994

Null mutations in the alpha PS2 and beta PS integrin subunit genes have distinct phenotypes.

The two Drosophila position-specific (PS) integrins are expressed on complementary sides of sites where different cell layers adhere to each other, such as the attachments of the embryonic muscles to the epidermis. While there is suggestive evidence that the PS integrin-mediated adhesion is via the extracellular matrix, it is also possible that it occurs through the direct interaction of the two integrins, alpha PS1 beta PS and alpha PS2 beta PS. To help distinguish between these possibilities a comparison between the phenotypes caused by the absence of the beta PS subunit and the absence of one of the PS alpha subunits, alpha PS2, has been made. Two pieces of evidence are provided that prove that the alpha PS2 subunit is encoded by the locus inflated (if). Firstly, three new if alleles have been isolated, each of which is associated with a molecular lesion in the alpha PS2 gene, and each of which results in the complete loss of if activity. Secondly, a 39 kb fragment of genomic DNA that encompasses the alpha PS2 gene completely rescues if mutations when introduced into the germline by P-element-mediated transformation. A comparison of the null inflated phenotype with that of the locus that encodes the beta PS subunit, myospheroid (mys), reveals that while the beta PS subunit is required for the adhesion of the epidermis along the dorsal midline, the alpha PS2 subunit is not. In if mutant embryos, the muscles remain attached to the other cell layers significantly longer than in a mys mutant embryo. This shows that the alpha PS2 beta PS integrin only contributes part of the adhesive activity at the sites of PS integrin adhesion, and rules out a model where PS integrin function occurs solely by the direct interaction of the two PS integrins.

Animals↗
PubMed1993

Integrins hold Drosophila together.

The Drosophila position-specific (PS) integrins are members of the integrin family of cell surface receptors and are thought to be receptors for extracellular matrix components. Each PS integrin consists of an alpha subunit, alpha PS1 or alpha PS2, and a beta PS subunit. Mutations in the beta PS subunit and the alpha PS2 subunit have been characterised and reveal that the PS integrins have an essential role in the adhesion of different cell layers to each other. The PS integrins are especially required for the function of the cell-matrix-cell junctions, where the muscles attach to the epidermis and where one surface of the developing wing adheres to the other. These junctions are similar to vertebrate focal adhesions and hemidesmosomes, which also contain integrins. Integrin-mediated cell to cell adhesion via the extracellular matrix provides a way for tissues to adhere to each other without intermingling of their cells.

Animals↗
PubMed1993

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth.

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1- mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1- mutants. Fe(II) transporter activity and regulation is normal in gef1- mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1- deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1- cells and by mitochondrial fractions isolated from gef1- mutants was reduced 25-50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

Amino Acid Sequence↗
PubMed1993

Integrins and morphogenesis.

The Drosophila position specific (PS) integrins consist of two cell surface heterodimers, PS1 (alpha PS1 beta PS) and PS2 (alpha OS2 beta PS), which are expressed on complementary sides of attachments between cell layers and are essential for these attachments. Current evidence suggests that the PS integrins bind to components of the extracellular matrix, similar to the majority of vertebrate integrins, but specific Drosophila ligands have not yet been identified. In the embryo PS1 is found on the surface of the epidermis and endoderm, while PS2 is restricted to the mesoderm. The integrins are concentrated at the sites where the somatic muscles attach to the epidermis and at the interface between the visceral mesoderm and the endoderm. In myospheroid mutant embryos, which lack the beta PS subunit, the adhesion between the mesoderm and the other cell layers fails. The PS integrins are also required for the adhesion of the dorsal to the ventral surface of the wing during metamorphosis. PS1 is expressed on the basal surface of the dorsal cells and PS2 is expressed on the ventral cells. Loss of PS integrin function in the wing results in balloon shaped wings because of the failure of the two surfaces of the wing blade to adhere to each other. These and other aspects of the phenotypes of mutations in the genes encoding the PS integrins indicate that integrins play an important role in the adhesion of different cell layers to each other and thus an essential role in the morphogenesis of the organism. The use of extracellular matrix receptors in this role may aid in keeping the different cell layers distinct.

Animals↗
PubMed1992

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.

Amino Acid Sequence↗