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 β subunit of the αβ spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. β 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 αβH spectrin and ankyrin were not significantly altered in β spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independently of one another, and that ankyrin is upstream of αβ spectrin in the spectrin assembly pathway. In contrast, β spectrin mutations had a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for β spectrin in determining the subcellular distribution of the Na,K ATPase and, unexpectedly, this role is independent of α spectrin.

Abstract. We show that the ot-spectrin gene is essential for larval survival and development by characterizing several ot-spectrin mutations in Drosophila.P-element minigene rescue and sequence analysis were used to identify the o~-spectrin gene as the l(3)dre3 complementatign group of the Dras-Roughenedecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an c~-speetrin eDNA, 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 -y-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 37-bp deletion in ot-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 mntation, 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.p HENOTYPIC analyses of spectrin deficiencies in mice and humans have provided strong evidence that the spectrin-based membrane skeleton of the erythrocyte contributes to the long range order and stability of the plasma membrane by enhancing mechanical integrity and deformability (Davies and Lux, 1989;Palek and Lambert, 1990). Despite a number of similarities in the structural properties of erythroid and non-erythroid spectrins, significant differences between the two suggest that non-erythroid spectrins may perform functions that are not predicted by the red blood cell model. In non-erythroid cells spectrin can and does interact with a far greater variety of proteins than exists in the erythrocyte (for a review see

Abstract. 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 $2 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. Doublelabeling 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.

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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.