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

Abstract

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.

Figure 1

Spectrin and ankyrin have distinct, partially overlapping distributions in neuroglian-expressing Drosophila tissue culture cells. Cell aggregates, formed upon expression of neuroglian, were double labeled with mouse anti-α spectrin (A) and rabbit antiankyrin (B) primary antibodies, followed by Texas red- and FITC-conjugated secondary antibodies, respectively. Bar, 10 μm.

Figure 3

Distinct distributions of two β spectrin isoforms in Drosophila tissue culture cells before and after expression of neuroglian and formation of cell–cell contacts. Control S2 cells (A and C) and neuroglian-expressing S2 cell aggregates (B and D) were attached to microscope slides and stained with antibodies against β spectrin (A and B; 1:500 serum) or β_H spectrin (C and D; 5 μg/ml) followed by FITC-conjugated secondary antibody. Arrowhead marks the accumulation of β spectrin at a cell–cell contact. Bar, 10 μm.

Figure 2

Western blot analysis of α spectrin and isoform-specific β spectrin antibodies. Total S2 cell proteins were reacted with monoclonal anti-α spectrin antibody (lane 1), polyclonal anti-β spectrin (lane 2), or affinity-purified anti-β_H spectrin antibodies (lane 3) and stained with alkaline phosphatase-conjugated secondary antibody.

Figure 4

The Na,K-ATPase codistributes with ankyrin at sites of neuroglian-mediated cell–cell contact. Neuroglian-expressing S2 cells were double labeled with mouse monoclonal anti-Na,K-ATPase antibody and rabbit antiankyrin antibody and secondary antibodies as in Figure 1. Bar, 10 μm.

Figure 5

Distribution of neuroglian, ankyrin, Na,K-ATPase, and β_H spectrin in the larval salivary gland epithelium. Salivary glands were dissected from late third instar wild-type larvae, fixed, permeabilized and double labeled with mouse antineuroglian (A), rabbit anti-Drosophila ankyrin (B, D, and F), mouse anti-Na,K-ATPase (C, E, and G); and rabbit anti-Drosophila β_H spectrin (H). Mouse antibodies were detected with Texas Red-conjugated secondary antibodies (A, C, E, and G) and rabbit antibodies were detected with FITC-conjugated secondary antibodies (B, D, F, and H). Neuroglian, ankyrin, and Na,K-ATPase colocalize at lateral sites of cell–cell contact (A–D). Ankyrin and the Na,K-ATPase colocalize at the lateral margin of salivary gland cells (arrow, E and F). Ankyrin is also concentrated at sites of cell adhesion (*, F) in the fat body (fb). Although the Na,K-ATPase was concentrated at lateral sites of cell contact in the salivary gland (thin arrow, G), the β_H subunit of spectrin was concentrated at the apical cell surface (wide arrow, H). Bars: A–D and G–H, 100 μM; E and F, 67 μm.

Figure 6

Distribution of ankyrin (A and C), β_H spectrin (B), and neuroglian (D) in the somatic follicle epithelium of the adult ovary. Ovaries were dissected from adult Drosophila females, fixed, permeabilized, and stained as in Figure 5. Arrows mark staining of ankyrin at lateral cell contacts (A and C), neuroglian at cell contacts (D), and β_H spectrin at the apical surface (B). Bar, 20 μm.

Figure 7

Segregation of spectrin isoforms in response to positional cues at the cell surface. (A) The uniform distribution of αβ_H spectrin (submembrane shading) at the plasma membrane of S2 cells (left) implies the presence of a constitutive β_H-specific receptor that is uniformly distributed at the cell surface. In contrast, ankyrin and αβ spectrin are found in the cytoplasm of control S2 cells (*, left), but they are recruited to sites of cell–cell contact in response to neuroglian-mediated cell adhesion (right). The Na,K-ATPase also becomes concentrated at cell contacts in response to neuroglian expression and ankyrin/αβ spectrin assembly. (B) Neuroglian, ankyrin, αβ spectrin, and Na,K-ATPase are concentrated at sites of cell–cell contact in the larval salivary gland epithelium, in agreement with the order of events observed in S2 cells. However, the putative αβ_H spectrin receptor is sorted apically in these polarized cells. Despite their common features (including an identical α subunit), the two spectrin isoforms respond independently to their respective membrane receptors to form segregated membrane skeleton domains.