Genetic studies of spectrin in the larval fat body of Drosophila melanogaster: evidence for a novel lipid uptake apparatus.

Spectrin cytoskeleton defects produce a host of phenotypes affecting the plasma membrane, cell polarity, and secretory membrane traffic. However, many of the underlying molecular mechanisms remain unexplained by prevailing models. Here we used the larval fat body of Drosophila melanogaster as a genetic model system to further elucidate mechanisms of αß-spectrin function. The results provide unexpected new insights into spectrin function as well as mechanisms of dietary fat uptake and storage. We show that loss of α- or ß-spectrin in the fat body eliminated a population of small cortical lipid droplets and altered plasma membrane architecture, but did not affect viability of the organism. We present a novel model in which αß-spectrin directly couples lipid uptake at the plasma membrane to lipid droplet growth in the cytoplasm. In contrast, strong overexpression of ß-spectrin caused fat body atrophy and larval lethality. Overexpression of ß-spectrin also perturbed transport of dietary fat from the midgut to the fat body. This hypermorphic phenotype appears to be the result of blocking secretion of the lipid carrier lipophorin from fat cells. However, this midgut phenotype was never seen with spectrin loss of function, suggesting that spectrin is not normally required for lipophorin secretion or function. The ß-spectrin hypermorphic phenotype was ameliorated by co-overexpression of α-spectrin. Based on the overexpression results here, we propose that ß-spectrin family members may be prone to hypermorphic effects (including effects on secretion) if their activity is not properly regulated.

The Drosophila Anion Exchanger (DAE) lacks a detectable interaction with the spectrin cytoskeleton.

BACKGROUND: Current models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1) was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE) as a marker for studies of the downstream effects of spectrin cytoskeleton mutations. RESULTS: Sequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila. CONCLUSIONS: Based on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

Transgene rescue identifies an essential function for Drosophila beta spectrin in the nervous system and a selective requirement for ankyrin-2-binding activity.

The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential beta spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a beta spectrin transgene or to knock down beta spectrin expression with dsRNA. The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation. Thus beta spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.