Ronald R. Dubreuil

Differential effects of a labial mutation on the development, structure, and function of stomach acid-secreting cells in Drosophila melanogaster larvae and adults

Abstract. The differentiation of copper cells, which secrete stomach acid in Drosophila larvae, has been shown previously to be sensitive to the labialk3 mutation. Here we found that stomach acid secretion in adults was insensitive to labk3. The basis for this stage-specific effect was elucidated by characterizing the development, structure, and function of the adult midgut. First, we demonstrated by copper-dependent fluorescence and morphology that copper cells were present in the adult stomach. Fine-structure analysis of adult copper cells led to the identification of a previously unrecognized plasma membrane domain: apicolateral contacts between copper cells and their neighbors consisted of smooth septate junctions that were enriched in αβ-spectrin and ankyrin. Second, we demonstrated that adult copper cells were present in labk3/labvd1 (conditional/null) adults. The labial protein was expressed in adult labk3/labvd1 copper cells, but not in larvae. Thus the labk3 mutation had a stage-specific effect on midgut labial expression, but did not appear to affect protein function. Surprisingly, stomach acidification was dispensable during larval development, since labk3/labvd1 mutant larvae that lacked midgut acidification developed into fertile adults.

β-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS

α- and β-Spectrin are major components of a submembrane cytoskeletal network connecting actin filaments to integral plasma membrane proteins. Besides its structural role in red blood cells, the Spectrin network is thought to function in non-erythroid cells during protein targeting and membrane domain formation. Here, we demonstrate that β-Spectrin is required in neurons for proper midline axon guidance in the Drosophila embryonic CNS. In β-spectrin mutants many axons inappropriately cross the CNS midline, suggesting a role forβ -Spectrin in midline repulsion. Surprisingly, neither the Ankyrin-binding nor the pleckstrin homology (PH) domains of β-Spectrin are required for accurate guidance decisions. α-Spectrin is dependent upon β-Spectrin for its normal subcellular localization and/or maintenance, whereas α-spectrin mutants exhibit a redistribution of β-Spectrin to the axon scaffold.β -spectrin mutants show specific dose-dependent genetic interactions with the midline repellent slit and its neuronal receptor roundabout (robo), but not with other guidance molecules. The results suggest that β-Spectrin contributes to midline repulsion through the regulation of Slit-Robo pathway components. We propose that the Spectrin network is playing a role independently of Ankyrin in the establishment and/or maintenance of specialized membrane domains containing guidance molecules that ensure the fidelity of axon repulsion at the midline.

Genetic studies of spectrin: new life for a ghost protein

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (beta H) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms. Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the beta H spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones.

Unexpected Complexity in the Mechanisms That Target Assembly of the Spectrin Cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant β-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP2) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP2 interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP2 and the spectrin PH domain codistribute with one another in cells but not with authentic wild type αβ-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP2 binding activity.

Genetic analysis of the requirements for α-actinin function

Null α-actinin mutations in Drosophila are lethal and produce conspicuous defects in muscle structure and function. Here, we used transgene rescue to examine the requirements for α-actinin function in vivo. First, we tested the ability of a cDNA-based transgene encoding the adult muscle isoform of α-actinin under control of the heterologous ubiquitin promoter to rescue the lethality of null α-actinin mutations. Successful rescue indicated that alternative splicing, which also generates larval muscle and non-muscle isoforms, was not essential for viability and that there were no strict spatial or temporal requirements for α-actinin expression. Secondly, chimeric transgenes, with functional domains of α-actinin replaced by similar domains from spectrin, were tested for their ability to rescue α-actinin mutants. Replacement of either the actin binding domain or the EF hand calcium binding domain yielded inactive proteins, indicating that these conserved domains were not functionally equivalent. Thirdly, the length of α-actinin was modified by adding a 114 amino acid structural repeat from α-spectrin to the center of the rod domain of α-actinin. Addition of this sequence module was expected to increase the length of the native α-actinin molecule by at least 15%, yet was fully compatible with α-actinin function as measured by rescued lethality and flight. Thus, unexpectedly, the exact length of α-actinin was not critical to its function in the muscle Z disk.

Chapter 8 Molecular and Genetic Dissection of the Membrane Skeleton in Drosophila

Publisher Summary This chapter describes a molecular and genetic dissection of the membrane skeleton in polarized cells in Drosophila . An important principle to emerge from the erythrocyte model is that the lipid bilayer is fortified by a submembrane network of peripheral membrane proteins. These proteins, commonly referred to as the “membrane skeleton,” are essential for the normal shape and stability of the erythrocyte membrane. The membrane-skeleton proteins first described in the erythrocyte also have homologs associated with the plasma membrane (PM) of other cell types. Biochemical studies have revealed that these homologs participate in the same molecular interactions as their erythrocyte counterparts. However, technical limitations have prevented a direct electron microscopic (EM) analysis of the membrane skeleton in nonerythroid cells. Genetic studies of the nonerythroid membrane skeleton have also been lacking. Consequently, much of the conceptual framework for membrane-skeleton function is borrowed from the erythrocyte model.