Engineering of III-Nitride Semiconductors on Low Temperature Co-fired Ceramics.

This work presents results in the field of advanced substrate solutions in order to achieve high crystalline quality group-III nitrides based heterostructures for high frequency and power devices or for sensor applications. With that objective, Low Temperature Co-fired Ceramics has been used, as a non-crystalline substrate. Structures like these have never been developed before, and for economic reasons will represent a groundbreaking material in these fields of Electronic. In this sense, the report presents the characterization through various techniques of three series of specimens where GaN was deposited on this ceramic composite, using different buffer layers, and a singular metal-organic chemical vapor deposition related technique for low temperature deposition. Other single crystalline ceramic-based templates were also utilized as substrate materials, for comparison purposes.

Differential effects of a labial mutation on the development, structure, and function of stomach acid-secreting cells in Drosophila melanogaster larvae and adults.

The differentiation of copper cells, which secrete stomach acid in Drosophila larvae, has been shown previously to be sensitive to the labialk3 mutation. Here we found that stomach acid secretion in adults was insensitive to labk3. The basis for this stage-specific effect was elucidated by characterizing the development, structure, and function of the adult midgut. First, we demonstrated by copper-dependent fluorescence and morphology that copper cells were present in the adult stomach. Fine-structure analysis of adult copper cells led to the identification of a previously unrecognized plasma membrane domain: apicolateral contacts between copper cells and their neighbors consisted of smooth septate junctions that were enriched in alphabeta-spectrin and ankyrin. Second, we demonstrated that adult copper cells were present in labk3/labvd1 (conditional/null) adults. The labial protein was expressed in adult labk3/labvd1 copper cells, but not in larvae. Thus the labk3 mutation had a stage-specific effect on midgut labial expression, but did not appear to affect protein function. Surprisingly, stomach acidification was dispensable during larval development, since labk3/labvd1 mutant larvae that lacked midgut acidification developed into fertile adults.

Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach.

Copper cells were originally identified in Drosophila midgut epithelium by their striking orange fluorescence in copper-fed larvae. Here, we examined copper cell fluorescence in light of the previous observations that (1) a similar fluorescent signal in yeast is produced by a complex between copper and metallothionein, and (2) metallothionein is expressed constitutively in the copper cell region and inducibly in other regions of the Drosophila midgut. Pulse-feeding experiments with 1 mM CuCl2 revealed that fluorescence appeared rapidly in copper cells (<5 min) and slowly in other cells of the midgut (days), suggesting a constitutive cofactor in the former and an inducible cofactor in the latter. Fluorescence was also detected in Drosophila S2 tissue culture cells after induction of metallothionein synthesis by addition of CuCl2 to the growth medium. Thus, fluorescence coincided spatially and temporally with the expression of metallothionein. Fluorescence was also linked to the acid-secreting activity of copper cells. Fluorescence was not observed when acid secretion was inhibited by a mutation in the alpha spectrin gene and acidification was blocked in copper-fed wild-type larvae. However, acidification was restored after a 1-day chase period in which the fluorescent signal became sequestered within a vesicular compartment. We therefore conclude that copper cell fluorescence is most probably attributable to a cytoplasmic copper-metallothionein complex, suggesting an unanticipated role for metallothionein in acid-secreting cells.

Drosophila alpha- and beta-spectrin mutations disrupt presynaptic neurotransmitter release.

Spectrins are plasma membrane-associated cytoskeletal proteins implicated in several aspects of synaptic development and function, including presynaptic vesicle tethering and postsynaptic receptor aggregation. To test these hypotheses, we characterized Drosophila mutants lacking either alpha- or beta-spectrin. The Drosophila genome contains only one alpha-spectrin and one conventional beta-spectrin gene, making it an ideal system to genetically manipulate spectrin levels and examine the resulting synaptic alterations. Both spectrin proteins are strongly expressed in the Drosophila neuromusculature and highly enriched at the glutamatergic neuromuscular junction. Protein null alpha- and beta-spectrin mutants are embryonic lethal and display severely disrupted neurotransmission without altered morphological synaptogenesis. Contrary to current models, the absence of spectrins does not alter postsynaptic glutamate receptor field function or the ultrastructural localization of presynaptic vesicles. However, the subcellular localization of numerous synaptic proteins is disrupted, suggesting that the defects in presynaptic neurotransmitter release may be attributable to inappropriate assembly, transport, or localization of proteins required for synaptic function.

Receptor clustering drives polarized assembly of ankyrin.

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.

Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells.

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. Here we used a genetic approach to directly test the requirement for the beta subunit of the alphabeta spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. beta Spectrin mutations were lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphabeta(H) spectrin and ankyrin were not significantly altered in beta spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independently of one another, and that ankyrin is upstream of alphabeta spectrin in the spectrin assembly pathway. In contrast, beta spectrin mutations had a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for beta spectrin in determining the subcellular distribution of the Na, K ATPase and, unexpectedly, this role is independent of alpha spectrin.

Genetic analysis of the requirements for alpha-actinin function.

Null alpha-actinin mutations in Drosophila are lethal and produce conspicuous defects in muscle structure and function. Here, we used transgene rescue to examine the requirements for alpha-actinin function in vivo. First, we tested the ability of a cDNA-based transgene encoding the adult muscle isoform of alpha-actinin under control of the heterologous ubiquitin promoter to rescue the lethality of null alpha-actinin mutations. Successful rescue indicated that alternative splicing, which also generates larval muscle and non-muscle isoforms, was not essential for viability and that there were no strict spatial or temporal requirements for alpha-actinin expression. Secondly, chimeric transgenes, with functional domains of alpha-actinin replaced by similar domains from spectrin, were tested for their ability to rescue alpha-actinin mutants. Replacement of either the actin binding domain or the EF hand calcium binding domain yielded inactive proteins, indicating that these conserved domains were not functionally equivalent. Thirdly, the length of alpha-actinin was modified by adding a 114 amino acid structural repeat from alpha-spectrin to the center of the rod domain of alpha-actinin. Addition of this sequence module was expected to increase the length of the native alpha-actinin molecule by at least 15%. yet was fully compatible with alpha-actinin function as measured by rescued lethality and flight. Thus, unexpectedly, the exact length of alpha-actinin was not critical to its function in the muscle Z disk.

Neuroglian and DE-cadherin activate independent cytoskeleton assembly pathways in Drosophila S2 cells.

The cytoskeletal proteins spectrin and ankyrin colocalize with sites of E-cadherin-mediated cell-cell adhesion in mammalian cells. Here we examined the effects of Drosophila DE-cadherin expression on spectrin and ankyrin in Drosophila S2 tissue culture cells. DE-cadherin caused a dramatic change in the cytoplasmic concentration and distribution of armadillo, the Drosophila homolog of beta catenin. However, DE-cadherin expression had no detectable effect on the quantity or subcellular distribution of ankyrin or spectrin. In reciprocal experiments, recruitment of ankyrin and alphabeta spectrin to the plasma membrane by another cell adhesion molecule, neuroglian, had no effect on the quantity or distribution of armadillo. The results indicate that DE-cadherin-catenin complexes and neuroglian-spectrin/ankyrin complexes form by nonintersecting pathways. Recruitment of spectrin does not appear to be a conserved feature of DE-cadherin function.

Structural requirements for outside-in and inside-out signaling by Drosophila neuroglian, a member of the L1 family of cell adhesion molecules.

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.

A conserved role for L1 as a transmembrane link between neuronal adhesion and membrane cytoskeleton assembly.

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, we demonstrate here that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations results from a disruption of the link between the extracellular environment and the neuronal cytoskeleton.

Mutations of alpha spectrin and labial block cuprophilic cell differentiation and acid secretion in the middle midgut of Drosophila larvae.

Mutations in Drosophila alpha spectrin cause larval lethality and defects in cell shape and adhesion (J. Lee et al., 1993, J. Cell Biol. 123, 1797-1809). Here we examined the effects of two lethal alpha spectrin alleles (alpha-specrg41 and alpha-specrg35) on development and function of the larval midgut. Homozygous null alpha-specrg41-mutant larvae exhibited a striking defect in middle midgut acidification. In contrast, many homozygous alpha-specrg35 mutants were capable of acidification, indicating partial function of the truncated alpha-specrg35 product. Acidification was also blocked by a mutation in the labial gene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. We found that two isoforms of spectrin (alphabeta and alphabetaH) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labial and alpha spectrin mutants was in morphogenesis of the invaginated apical domain, although basolateral defects may also contribute to the acidification phenotype. Acid secretion in vertebrate systems is thought to involve the polarized activities of apical proton pumps and basolateral anion exchangers, both of which interact with spectrin. We propose that the alpha-specrg41 mutation in Drosophila interferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells.

Genetic studies of spectrin: new life for a ghost protein.

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (beta H) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms. Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the beta H spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones.

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Ethanol does not inhibit the adhesive activity of Drosophila neuroglian or human L1 in Drosophila S2 tissue culture cells.

Members of the L1 family of homophilic neural cell adhesion molecules are thought to play an important role in nervous system development and function. It is also suggested that L1 is a direct target of ethanol in fetal alcohol syndrome, since ethanol inhibits the aggregation of cultured cells expressing L1 (Ramanathan, R., Wilkemeyer, M. F., Mittel, B., Perides, G., and Charness, M. E. (1996) J. Cell Biol. 133, 381-390). If ethanol acts directly on the homophilic adhesive function of the L1 molecule, then inhibition of aggregation by ethanol should be observed in any cell type that expresses L1. Here we examined the effect of physiologically relevant concentrations of ethanol on the aggregation of Drosophila S2 cells that expressed either neuroglian (the Drosophila homolog of L1) or human L1. The aggregation of these S2 cells is known to be solely dependent on the homophilic interactions between L1 or neuroglian molecules. Neither cell adhesion molecule was affected when cell aggregation assays were carried out in the presence of >/=38 mM ethanol. The recruitment of membrane skeleton assembly at sites of cell-cell contact (a transmembrane signaling function of human L1) was also unaffected by the presence of ethanol. Thus the previously described inhibition of cell adhesion by ethanol in L1-expressing cells cannot be explained by a simple direct effect on the adhesive activity of L1 family members.

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites.

The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.

Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila.

We report the identification and initial characterization of Drosophila melanogaster ankyrin. Oligonucleotide primers based on the spectrin-binding domain of human brain ankyrin were used to amplify Drosophila genomic DNA. A cloned 184-bp PCR product was used to isolate Drosophila ankyrin cDNAs. Ankyrin cDNA probes detected a 5.5-kb transcript from embryonic poly(A)+ RNA and a single polytene chromosome locus (101F-102A). The cDNA sequence encodes a 170-kDa protein that is 53% identical to human brain ankyrin (Ank2). Antibodies directed against a recombinant fragment of Drosophila ankyrin reacted with a 170-kDa polypeptide from Drosophila embryos, larvae, S2 cells, and adult flies. The ankyrin antibody coimmunoprecipitated alpha- and beta-spectrin with ankyrin in detergent extracts of Drosophila embryo membranes. Antibodies against Drosophila ankyrin, alpha-spectrin and beta-spectrin were used to detect these proteins in wild-type and alpha-spectrin-mutant larvae. alpha-Spectrin levels were greatly diminished in mutant larvae, but levels of ankyrin and beta-spectrin were indistinguishable from wild type. The persistence of ankyrin and beta-spectrin may explain the relatively mild phenotype of alpha-spectrin mutants during early Drosophila development.

Monoclonal antibodies directed against human tumor-associated antigens cross-react with Drosophila proteins in clusters.

We reacted the Third International IASLC Workshop panel of monoclonal antibodies (MAbs) directed against human lung-tumor-associated epitopes with nitrocellulose blots of total proteins from adult fruit flies. Out of 63 MAbs tested, 9 showed a significant reaction with Drosophila proteins. Interestingly, in a double-blind analysis, most of the positive reactions fell into clusters that parallel the antibody reactivities against human tissues. In light of our findings, it becomes possible to screen expression vector libraries in order to isolate Drosophila cDNA that may have homology with human tumor-associated antigens.

Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster.

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.

Structure and evolution of the actin crosslinking proteins.

The actin crosslinking proteins exhibit marked diversity in size and shape and crosslink actin filaments in different ways. Amino acid sequence analysis of many of these proteins has provided clues to the origin of their diversity. Spectrin, alpha-actinin, ABP-120, ABP-280, fimbrin, and dystrophin share a homologous sequence segment that is implicated as the common actin binding domain. The remainder of each protein consists of repetitive and non-repetitive sequence segments that have been shuffled and multiplied in evolution to produce a variety of proteins that are related in function and in composition, but that differ significantly in structure.