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

Abstract

SummaryCell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner  Heavy -spectrin ( H ), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of  H (H33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that  H coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.Key words: Drosophila, Cell adhesion, Apoptosis, Spectrin, Membrane skeleton, Crumbs, IrreC, Roughest Journal of Cell Science 278Phillips and Thomas, 2006;Stabach et al., 2009) activities, spectrins can variously stabilize proteins at the membrane, nucleate protein complexes and modulate protein delivery and recycling. The Crumbs- H complex has been implicated in the development and maintenance of the apical domain via the modulation of endocytosis in both lossof-function and gain-of-function experiments (Pellikka et al., 2002;Williams et al., 2004). This activity appears to be centered on the Cterminal segment 33 of  H (H33), the overexpression of which causes membrane growth and the sequestration of Dynamin (Williams et al., 2004). In some tissues, expression of H33 also induces apoptosis (Williams et al., 2004). These phenotypes are  H specific, because overexpression of the equivalent region of the basolateral -spectrin does not have this effect (Williams et al., 2004). Modulation of endocytosis or recycling of junctional proteins through interactions with segment 33 of  H might thus provide the normal mechanism whereby the ZA is stabilized.Here, we further characterize the effects of H33 in the developing eye. We find that H33 expression preferentially results in the loss of IOC. We further show that these defects in morphogenesis are associated with fragmentation of the ZA and disruption of the Ig-CAM Roughest. In addition, we show that  H genetically and physically interacts with Roughest to maintain its wild-type distribution. Because  H also supports the ZA, we suggest that the ac...

Annexin B9 binds to βH-spectrin and is required for multivesicular body function in Drosophila

“…b H is conspicuously associated with the ZA (Lee et al, 2010;Thomas and Kiehart, 1994;Thomas et al, 1998;Zarnescu and Thomas, 1999) and is required for its integrity (Zarnescu and Thomas, 1999). Given the growing association of b H with protein recycling (Phillips and Thomas, 2006;Williams et al, 2004) (this paper), we hypothesize that this phenotype arises from defective or inappropriate trafficking of DE-Cadherin.…”

“…Roles for the SBMS have been demonstrated in multiple organisms and tissues including: in neuronal structure, function and membrane organization (Hammarlund et al, 2007;Hulsmeier et al, 2007;Ikeda et al, 2006;Lacas-Gervais et al, 2004;Pielage et al, 2006); in epithelial structure and stability (Lee et al, 2010;Lee et al, 1997;Thomas et al, 1998); and in muscle function (Bennett and Healy, 2008;Mohler et al, 2005). Widely regarded as a static structural element, emerging results have indicated that these proteins actually have dynamic roles in transport processes including: trans-Golgi network to plasma membrane (Kizhatil et al, 2007a); ER to Golgi (Stabach et al, 2008;Stankewich et al, 2010); at the early endosome (Phillips and Thomas, 2006); and endosome to lysosome transport (Johansson et al, 2007).…”

“…b Heavy -spectrin (b H ) is apically restricted in most tissues (Thomas and Kiehart, 1994) and is required for epithelial morphogenesis (Lee et al, 2010;Thomas et al, 1998;Zarnescu and Thomas, 1999). In primary epithelia, b H is recruited by the apical polarity determinant Crumbs (Medina et al, 2002;Pellikka et al, 2002) through the FERM-binding motif in the Crumbs cytoplasmic domain (Medina et al, 2002).…”

“…In primary epithelia, b H is recruited by the apical polarity determinant Crumbs (Medina et al, 2002;Pellikka et al, 2002) through the FERM-binding motif in the Crumbs cytoplasmic domain (Medina et al, 2002). This motif is required to stabilize the Cadherin-based zonula adherens (ZA) (Klebes and Knust, 2000), and loss-of-function mutations in karst (which encodes b H ) result in a mild disruption of the ZA (Zarnescu and Thomas, 1999) and of the Ig-CAM, Roughest (Lee et al, 2010). This disruption probably results in the morphogenetic defects seen in karst mutants; however, the specific role played by b H in stabilizing these junctions remains unknown.…”

Spectrin βV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians

“…3B), whereas the Drosophila spectrin β H preferentially interacts with PI(4,5)P2 (24). This could partially account for the targeting of these unconventional β spectrins to different cell regions or compartments.…”