The C-terminal domain ofDrosophilaβHeavy-spectrin exhibits autonomous membrane association and modulates membrane area

Abstract

Current models of cell polarity invoke asymmetric cues that reorganize the secretory apparatus to induce polarized protein delivery. An important step in this process is the stabilization of the protein composition in each polarized membrane domain. The spectrin-based membrane skeleton is thought to contribute to such stabilization by increasing the half-life of many proteins at the cell surface. Genetic evidence is consistent with a negative role for Drosophila βHeavy-spectrin in endocytosis, but the inhibitory mechanism has not been elucidated. Here, we investigated the membrane binding properties of the C-terminal nonrepetitive domain of βHeavy-spectrin through its in vivo expression in transgenic flies. We found that this region is a membrane-association domain that requires a pleckstrin homology domain for full activity, and we showed for the first time that robust membrane binding by such a C-terminal domain requires additional contributions outside the pleckstrin homology. In addition, we showed that expression of the βHeavy-spectrin C-terminal domain has a potent effect on epithelial morphogenesis. This effect is associated with its ability to induce an expansion in plasma membrane surface area. The membrane expansions adopt a very specific bi-membrane structure that sequesters both the C-terminal domain and the endocytic protein dynamin. Our data provide supporting evidence for the inhibition of endocytosis by βHeavy-spectrin, and suggest that the C-terminal domain mediates this effect through interaction with the endocytic machinery. Spectrin may be an active partner in the stabilization of polarized membrane domains.

The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila

“…This is consistent with previous observations that the distribution of Roughest is dependent on the integrity of the ZA (Fig. 5D-I) (Grzeschik and Knust, 2005) and vice versa (Bao and Cagan, 2005).We next localized H33 and  H in GMR>H33 flies via its Myc tag (Williams et al, 2004). H33 exhibits an unpolarized membrane distribution at all stages in all cell types (Fig.…”

“…Deletion of the PH domain prevents both phenotypes ( Fig. 1) (Williams et al, 2004). We compared the lipid-binding specificity of the  H PH domain with the closely related PH domain from -spectrin, which does not have these dominant effects.…”

“…A construct comprising regions i, ii and iii, inhibits endocytosis and induces apoptosis. A smaller construct comprising regions ii and iii induces only apoptosis (see Williams et al, 2004). …”

“…We have found that the overexpression of this domain disrupts development, is associated with acridine orange accumulation and can be ameliorated by coexpression of the baculovirus p35 caspase inhibitor, revealing it to be at least in part apoptotic (Williams et al, 2004). This is a specific effect of the apical  H isoform, because overexpression of the C-terminus of the related basolateral -spectrin produces no such phenotype (Williams et al, 2004).H33 contains a PH domain, which is required to induce this phenotype (Williams et al, 2004), indicating a requirement for lipid binding or overlapping protein binding (e.g. Touhara et al, 1995).…”

αII-βV spectrin bridges the plasma membrane and cortical lattice in the lateral wall of the auditory outer hair cells

“…The fact that Drosophila β-heavy spectrin (Williams et al, 2004), Caenorhabditis elegans Sma-1 (McKeown et al, 1998) and mammalian βV spectrin each have independently maintained a 30-repeat length throughout evolution (Stabach and Morrow, 2000), argues in favor of a crucial role for either a longer actin-membrane cross-linker or the need for greater extensible flexibility than can be provided by the other smaller conventional spectrins. The unusually large size of the βV spectrin subunit, and the link between this spectrin and the molecular motor prestin, highlights the need to integrate these data into current models of the OHC electromotile behavior in response to sound stimulation.…”