Michael P. Krahn

Membrane targeting of Bazooka/PAR-3 is mediated by direct binding to phosphoinositide lipids.

Cell polarity in higher animals is controlled by evolutionarily conserved protein complexes, which localize to the cytocortex in a polarized manner. The PAR-3/PAR-6/atypical protein kinase C (aPKC) complex is the first to become asymmetrically localized, and it controls the localization of additional complexes functioning further downstream in the regulation of cell polarity. The first component of the PAR-3/PAR-6/aPKC complex that is localized to the cortex is Bazooka/PAR-3 (Baz), a large scaffolding protein. In most cell types analyzed, loss of Baz function leads to loss of cell polarity. Here we present a structure-function analysis of Baz focusing on its subcellular localization and function in four different polarized Drosophila cell types: the embryonic ectodermal epidermis, the follicular epithelium, embryonic neuroblasts, and the oocyte. We show that the PDZ domains of Baz are dispensable for its correct localization, whereas a conserved region in the C-terminal part of Baz to which no function had been assigned so far is required and sufficient for membrane localization. This region binds to phosphoinositide membrane lipids and thus mediates cortical localization of Baz by direct interaction with the plasma membrane. Our findings reveal a mechanism for the coupling of plasma membrane polarity and cortical polarity.

Formation of a Bazooka-Stardust complex is essential for plasma membrane polarity in epithelia.

Recruitment of the Crumbs–Stardust polarity complex depends on interactions between Bazooka and the Stardust PDZ domain and is regulated by aPKC-mediated phosphorylation.

Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity.

PATJ indirectly promotes apical–basal polarity in epithelial cells by enhancing Myosin phosphorylation and thereby stabilizing adherens junctions.

Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex

Many cell types in animals and plants are polarized, which means that the cell is subdivided into functionally and structurally distinct compartments. Epithelial cells, for example, possess an apical side facing a lumen or the outside environment and a basolateral side facing adjacent epithelial cells and the basement membrane. Neurons possess distinct axonal and dendritic compartments with specific functions in sending and receiving signals. Migrating cells form a leading edge that actively engages in pathfinding and cell-substrate attachment, and a trailing edge where such attachments are abandoned. In all of these cases, both the plasma membrane and the cytocortex directly underneath the plasma membrane show differences in their molecular composition and structural organization. In this chapter we will focus on a specific type of membrane lipids, the phosphoinositides, because in polarized cells they show a polarized distribution in the plasma membrane. They furthermore influence the molecular organization of the cytocortex by recruiting specific protein binding partners which are involved in the regulation of the cytoskeleton and in signal transduction cascades that control polarity, growth and cell migration.

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz4 and baz815-8 alleles with those of the so far uncharacterized bazXR11 and bazEH747 null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in bazEH747 and bazXR11 while baz4 and baz815-8 show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz4 and baz815-8 alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type.

Src kinases mediate the interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase signalling efficiency in Drosophila ectodermal cells

Intercellular communication depends on the correct organization of the signal transduction complexes. In many signalling pathways, the mechanisms controlling the overall cell polarity also localize components of these pathways to different domains of the plasma membrane. In the Drosophila ectoderm, the JAK/STAT pathway components are highly polarized with apical localization of the receptor, the associated kinase and the STAT92E protein itself. The apical localization of STAT92E is independent of the receptor complex and is due to its direct association with the apical determining protein Bazooka (Baz). Here, we find that Baz-STAT92E interaction depends on the presence of the Drosophila Src kinases. In the absence of Src, STAT92E cannot bind to Baz in cells or in whole embryos, and this correlates with an impairment of JAK/STAT signalling function. We believe that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, as we can co-precipitate Src with Baz but not with STAT92E. This is the first time that a functional link between cell polarity, the JAK/STAT signalling pathway and the Src kinases has been established in a whole organism.

Notch Signaling: Linking Delta Endocytosis and Cell Polarity

Activation of Notch by its transmembrane ligand Delta requires the E3 ubiquitin ligases Neuralized or Mind bomb and endocytosis of the ubiquitinated ligand. In this issue of Developmental Cell, Ossipova et al. show that the polarity regulator PAR-1 phosphorylates Mind bomb, leading to the degradation of Mind bomb and to changes in cell fate due to loss of Notch signaling.

Die Funktion des PAR/aPKC-Komplexes in Drosophila

ZusammenfassungViele Zelltypen in mehrzelligen Organismen weisen eine ausgeprägte Polarität auf, die für die Funktion der Zellen von größter Bedeutung ist. Die Kontrolle der Zellpolarität erfolgt durch ein evolutionär hochkonserviertes Netzwerk interagierender Proteine.AbstractMany cell types in multicellular organisms possess a pronounced polarity that is of great importance for the function of the cells. Cell polarity is controlled by an evolutionarily conserved network of interacting proteins.

08-P018 The role of the Bazooka–CG31534 interaction for the establishment of cell polarity in Drosophila melanogaster

network. This work describes how the transcripts of several non-LTR retrotransposons reach the oocyte nucleus by using the same localisation machinery as endogenous RNAs such as gurken. Visualisation and analysis of I factor retrotransposon localisation reveals that its RNA localises more efficiently than the endogenous gurken transcript. We show here that these differences in localisation efficiency can be directly attributed to the physical properties of structural motifs within the I factor RNA and their effects on RNA–RNA interaction. Not only does the I factor have a localisation motif with 2D structural similarity to the gurken localisation motif but it also contains an adjacent sequence that enhances localisation by promoting ribonucleoprotein (RNP) particle assembly and recognition. We propose a general principle that RNP particle assembly and recognition represents a key regulatory point in the RNA localisation process.

08-P008 The poorly conserved C-terminus of Bazooka links the PAR complex to the apical membrane via a new lipid binding motif

Phosphorylation of Bazooka (Baz)/PAR-3 by atypical protein kinase C (aPKC) in Drosophila has been supposed to be a crucial event in the formation and functionality of the PAR/aPKC complex, stabilizing the complex at the apical membrane and subsequently recruiting other polarity markers or cell fate determinants to either the apical or basal membrane in epithelia or neural stem cells (neuroblasts, NBs). However, all these hypotheses are derived from the described interaction between mammalian PAR-3 and PKC. Although Baz and PAR-3 as well as the three major atypical PKC isoforms (iota, zeta and lambda) are highly conserved between fly and man, we reveal in this study remarkable differences. Baz binds aPKC not only by its conserved aPKC binding motif but also via its second and third PDZ domain. Deletion of the PDZ domains almost abolishes Baz binding to aPKC without affecting the phosphorylation at S980. We show that in contrast to its behaviour in a mammalian cell culture system, phosphorylation of Baz by aPKC at S980 does not lead to weakening of the complex but in contrast to a stronger binding. Furthermore, we demonstrate that impaired phosphorylation of Baz by replacement of serine 980 by alanine results in a dramatic dominant negative phenotype with total loss of polarity only in ectodermal epithelia, whereas in embryonic NBs, mesodermal follicle cells and the female germline, no defects can be observed.