Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate.

The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. Here, we identify phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. We propose that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side.

Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila.

The polarized architecture of epithelia relies on an interplay between the cytoskeleton, the trafficking machinery, and cell-cell and cell-matrix adhesion. Specifically, contact with the basement membrane (BM), an extracellular matrix underlying the basal side of epithelia, is important for cell polarity. However, little is known about how BM proteins themselves achieve a polarized distribution. In a genetic screen in the Drosophila follicular epithelium, we identified mutations in Crag, which encodes a conserved protein with domains implicated in membrane trafficking. Follicle cells mutant for Crag lose epithelial integrity and frequently become invasive. The loss of Crag leads to the anomalous accumulation of BM components on both sides of epithelial cells without directly affecting the distribution of apical or basolateral membrane proteins. This defect is not generally observed in mutants affecting epithelial integrity. We propose that Crag plays a unique role in organizing epithelial architecture by regulating the polarized secretion of BM proteins.

Differential effects of Cbl isoforms on Egfr signaling in Drosophila.

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.

Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline.

Cks is a small highly conserved protein that plays an important role in cell cycle control in different eukaryotes. Cks proteins have been implicated in entry into and exit from mitosis, by promoting Cyclin-dependent kinase (Cdk) activity on mitotic substrates. In yeast, Cks can promote exit from mitosis by transcriptional regulation of cell cycle regulators. Cks proteins have also been found to promote S-phase via an interaction with the SCF(Skp2) Ubiquitination complex. We have characterized the Drosophila Cks gene, Cks30A and we find that it is required for progression through female meiosis and the mitotic divisions of the early embryo through an interaction with Cdk1. Cks30A mutants are compromised for Cyclin A destruction, resulting in an arrest or delay at the metaphase/anaphase transition, both in female meiosis and in the early syncytial embryo. Cks30A appears to regulate Cyclin A levels through the activity of a female germline-specific anaphase-promoting complex, CDC20-Cortex. We also find that a second closely related Cks gene, Cks85A, plays a distinct, non-overlapping role in Drosophila, and the two genes cannot functionally replace each other.