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.

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.

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, calmodulin-binding, and calcium-binding properties of recombinant alpha spectrin polypeptides.

We examined the structure and the distribution of binding activities within bacterially produced fragments of Drosophila alpha spectrin. By electron microscopy, purified spectrin fragments resembled the corresponding regions of native spectrin. The contour lengths of recombinant spectrin molecules were proportional to the length of their coding sequences, which is consistent with current models of spectrin structure in which individual segments of the polypeptide contribute independently to the structure of the native molecule. We localized two sites at which calcium may regulate spectrin function. First, a site responsible for calmodulin binding to Drosophila alpha spectrin was identified near the junction of repetitive segments 14 and 15. Second, a domain of Drosophila alpha spectrin that includes two EF hand calcium-binding sequences bound 45Ca in blot overlay assays. EF hand sequences from a homologous domain of Drosophila alpha actinin did not bind calcium under the same conditions.

Properties and topography of the major integral plasma membrane protein of a unicellular organism.

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta-mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39-kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39-kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.

The membrane skeleton of a unicellular organism consists of bridged, articulating strips.

In this paper we show that a membrane skeleton associated with the plasma membrane of the unicellular organism Euglena consists of approximately 40 individual S-shaped strips that overlap along their lateral margins. The region of strip overlap is occupied by a set of microtubule-associated bridges and microtubule-independent bridges. Both cell form and plasma membrane organization are dependent on the integrity of this membrane skeleton. Removal of the membrane skeleton with a low-molar base results in loss of membrane form and randomization of the paracrystalline membrane interior characteristic of untreated cells. Conversely, removal of the plasma membrane and residual cytoplasm with lithium 3,5-diiodosalicylate/Nonidet P-40 yields cell ghosts that retain the form of the original cell but consist only of the membrane skeleton. Two major polypeptides of 86 and 80 KD persist in the skeleton and two other major proteins of 68 and 39 kD are associated with the plasma membrane fraction. None of these components appears to be the same as the major polypeptides (spectrins, band 3) of the erythrocyte ghost, the other cell system in which a well-defined peripheral membrane skeleton has been identified. We suggest that the articulating strips of euglenoids are not only the basic unit of cell and surface form, but that they are also positioned to mediate or accommodate surface movements by sliding, and to permit surface replication by intussusception.