Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P₂inDrosophila

Abstract

28An efficient vectorial intracellular transport machinery depends on a well-established apico-29 basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive 30 knowledge on individual trafficking pathways, little is known about the mechanisms 31 coordinating their temporal and spatial regulation. Here, we report that the polarity protein 32 Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient 33 apical secretion. Through recruiting Heavy-Spectrin and MyosinV to the apical membrane, 34 Crumbs maintains the Rab6-, Rab11-and Rab30-dependent trafficking and regulates the lipid 35 phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of 36 PI(4,5)P2 and formation of a novel, Moesin-and PI(4,5)P2-enriched apical membrane sac 37 containing microvilli-like structures. Our results identify Crumbs as an essential hub required 38 to maintain the organization of the apical membrane and the physiological activity of the larval 39 salivary gland. 40 41 Epithelia can organize as layers or tubes, which form barriers and thus separate 43 internal biological compartments from the environment. Many epithelia are specialized for 44 absorption or secretion by performing selective and directional transport of nutrients, enzymes 45 and waste products, which is essential for metazoan life (Cereijido et al., 2004; Rodriguez-46 Boulan and Macara, 2014; Lemaitre and Miguel-Aliaga, 2013). To perform these functions, 47 epithelial cells are highly polarized: plasma membrane proteins and lipids are distributed 48 asymmetrically into an apical domain facing the environment or a lumen, and a basolateral 49 domain that contacts the neighboring cell and/or a basal lamina. In addition, polarity is 50 manifested by uneven distribution of organelles, asymmetric cytoskeleton organization and 51 directed trafficking (Rodriguez-Boulan and Macara, 2014; Knust and Bossinger, 2002; Eaton 52 and Martin-Belmonte, 2014). The latter is particularly obvious in secretory epithelia, for 53 example the salivary glands, which produce vast amounts of material that is secreted into the 54 gland lumen (Blasky et al., 2015; Iruela-Arispe and Beitel, 2013; Eaton and Martin-Belmonte, 55 2014; Chung et al., 2014; Miguel-Aliaga et al., 2018).56 Several evolutionarily conserved proteins regulate epithelial cell polarity. These include 57 members of the apical Crumbs-and PAR-complexes, and the basolateral Scrib-Dlg-Lgl 58 module (reviewed in (Flores-Benitez and Knust, 2016; Roman-Fernandez and Bryant, 2016)). 59The Crumbs (Crb) protein has a large extracellular domain (>2000 aa), and a small intracellular 60 domain (37 aa) (Tepass et al., 1990; Wodarz et al., 1993), which harbors two protein-protein 61 interaction motifs, a C-terminal PDZ (Postsynaptic density/Discs large/ZO-1)-domain binding 62 motif (PBM) and a juxtamembrane FERM (protein 4.1/ezrin/radixin/moesin)-domain binding 63 motif (FBM). The PBM is important for cell polarity and can bind Stardust (Sdt) and Par-6 (Li

Progenitor cell integration into a barrier epithelium during adult organ turnover

“…Notably, Moesin, which outlines PAACs in confocal micrographs (Fig. 3 and Video 3), is a marker of microvilli in other Drosophila epithelia (Edwards et al, 1997;Lattner et al, 2019). We also found sausage-shaped (allantoid) ultrastructures that, like the ellipsoids, are lined with dense microvilli and circumscribed by SJs (Fig.…”

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma

“…In vivo, Syx is bound to the CRUMBS complex via Mupp-1 (6,74,75). Given the membrane trafficking at this site, it would make sense that the PH domain of this protein would likely bind PI(4,5)P2, the canonical ligand for PH domains(9, 76), because PI(4,5)P2 is also associated with actin cytoskeletal activity modulated by the CRUMBS polarity complex (77,78). Active RhoA stimulates PIP 5-kinase and subsequent formation of PI(4,5)P2, implicating a positive feedback mechanism where active RhoA results in production of PI(4,5)P2, which recruits Syx to reactivate RhoA as part of a cell's apical cell polarity program (79,80).…”