Cytogenetic map of the β spectrin region of the X chromosome. Three overlapping duplications define intervals of the X chromosome. 20 ethyl methane sulfonate–induced mutations in the β spectrin region were ordered into six complementation groups within these intervals (vertical rows). None of the interval II complementation groups complemented the X chromosome deficiency Df(1)SD10. Note that the order of complementation groups within intervals II and III is not known.

Relative distributions of β spectrin and ankyrin in copper cells. (A) Copper cell morphology. Copper cells alternate with interstitial cells in the larval middle midgut epithelium. The apical surface of the copper cell is invaginated and opens into the gut lumen through a pore formed by surrounding interstitial cells. The basolateral surface is spheroid in shape and completely surrounds the invaginated apical domain, except at the pore. The basolateral domain immediately surrounding the pore (apicolateral, arrow) forms a septate junction with the adjacent region of the interstitial cells. (B) Dissected middle midgut from a first instar larva expressing the myc epitope–tagged β spectrin transgene stained with mouse mAb against the epitope tag and Texas red–labeled goat anti–mouse secondary antibody. The most intense staining was found at the apicolateral domain of copper cells (arrowhead), with weaker staining of the rest of the basolateral plasma membrane (arrow). (C) Double labeling of the same sample as in A, stained with affinity-purified rabbit anti-ankyrin antibody and FITC-conjugated secondary antibody. Arrowhead marks comma-shaped staining of the apicolateral contacts between copper cells and neighboring interstitial cells in this optical section. (D) Staining of a second instar β-spec⁺ larva with ankyrin antibody as in C. Arrow marks staining of the basolateral domain of copper cells and arrowhead marks the relatively intense staining of the apicolateral contact region. Bar, 10 μM.

Localization of the Na,K ATPase in β spectrin mutants by confocal microscopy. Dissected preparations of the middle midgut from wild-type control larvae (A and B) and β-spec^em6 mutants (C and D) were stained with mouse monoclonal anti–Na,K ATPase α subunit and fluorescent secondary antibody. The basolateral staining pattern of the Na,K ATPase in wild-type appeared as rings in en face views (A), or as horseshoe shapes in optical sections near the center of the midgut (B). Neither pattern was detectable in the β-spec^em6 mutants (C and D). Toward the anterior (C and D, arrowhead), Na,K ATPase staining was distributed throughout the cytoplasm. Toward the posterior (C and D, arrow), staining appeared as large puncta and did not appear to be associated with the plasma membrane. In between these two regions, staining was relatively weak or in some cases absent. Bar, 10 μM.

Stomach acidification was analyzed by feeding β spectrin mutant larvae and their wild-type siblings with bromphenol blue–dyed yeast paste. Acidification was scored by examining the dye color in dissected midguts. Results are presented as the percentage of larvae with strong (yellow), moderate (green), or no detectable midgut acidification (blue).

(A) Western blot analysis of β spectrin mutant embryos. Mutant male embryos (lanes 2, 4, 6 and 8) and wild-type female siblings (lanes 1, 3, 5, and 7) were homogenized in SDS sample buffer and stained with rabbit anti–Drosophila β spectrin antibody after electrophoresis and transfer to nitrocellulose. Alkaline phosphatase–conjugated goat anti–rabbit antibody was used as secondary label. Mobilities of molecular weight standards are shown to the left in kD. An asterisk marks truncated products observed in two of the mutants. (B) Results of genomic sequence analysis from the β spectrin mutants is shown schematically. (+) The domain structure of wild-type β spectrin includes the actin binding domain (ABD), a series of ∼106 amino acid spectrin repeats, an ankyrin binding site, and a plekstrin homology region (PH). The β-spec^em12 mutation was caused by a nonsense mutation within the codon for a highly conserved tryptophan residue in repeat 12. The β-spec^em21 mutation was caused by a nonsense mutation at the same conserved tryptophan codon in repeat 13.

Localization of ankyrin in β spectrin mutants. Ankyrin staining (as in Fig. 3) was used to generate en face views of cell pattern in the posterior region of dissected wild-type (A) and β-spec^em6 mutant (B and C) middle midguts. Circular profiles (arrows) represent apicolateral contacts between copper cells and interstitial cells, which are consistently small in wild-type but large and irregular in mutants. Lines connecting circles (arrowhead) represent staining of apicolateral contact sites between neighboring interstitial cells. Bar, 10 μM.

Localization of α and β_H spectrin in β spectrin mutants by confocal microscopy. Dissected preparations of middle midgut were double-labeled with mouse monoclonal anti–α spectrin (A and D) and rabbit anti–β_H spectrin (B and E) and fluorescent secondary antibodies as in Fig. 3. The two subunits colocalized in the apical membrane domain of copper cells in wild-type (A–C, arrows) and in β-spec^em6 copper cells (D and E, arrows). Although α spectrin was most prominently visible at the basolateral surface of wild-type copper cells (A), staining of the apical surface predominated in the β spectrin mutants (D). In some cases, α and β_H spectrin colocalized at the basolateral surface of mutant copper cells (D and E, arrowheads). Bar, 20 μM.

Schematic models for the flow of positional information through the spectrin membrane skeleton. (a) The spectrin tetramer includes two ankyrin binding sites that provide potential interaction sites for a source of positional information (e.g., a cell adhesion molecule [CAM]) and for other membrane markers such as the Na,K ATPase. (b) Biochemical studies indicate that mammalian ankyrin can simultaneously interact with two different integral membrane proteins. The results shown here indicate that ankyrin alone, in the absence of β spectrin, is not sufficient to direct the normal basolateral accumulation of Na,K ATPase. β spectrin may indirectly affect the activity of ankyrin, perhaps through an allosteric mechanism. (c) Alternatively, β spectrin dimers may couple two ankyrin molecules that independently associate with a source and a target of positional information. One possible mechanism of β spectrin dimer formation is through its NH₂-terminal actin binding activity.