We focus on the so-called planar cell polarity (or in short: PCP) pathway that polarizes cells in the plane of a tissue. In epithelia, the PCP pathway acts perpendicular to the apico-basal pathway and also shows molecular interactions with apico-basal determinants such as aPKC, Par3 and Scribble. Genetic screens in Drosophila melanogaster have identified a set of conserved PCP core factors. Mutations in these factors lead to defects in wing hair patterning, sensory bristle orientation in the notum and abdomen, and ommatidial rotation in the eye. More recently, many vertebrate tissues and developmental processes have also been shown to display typical PCP features and to be regulated by the same core factors as those in Drosophila. Generally, tissues that develop cellular appendages such as inner ear sensory cells use PCP to organize their alignment. Furthermore, collective directional cell migration and tissue fusion processes, as found in gastrulation or in epidermal wound healing, respectively, are important PCP-dependent events. As a consequence, defective PCP signaling contributes to many diseases including neural tube defects or polycystic kidney disease. On the molecular level, there are two conserved PCP protein cassettes: the Fat/Dachsous group and the classical PCP core group consisting of Frizzled (Fz), Dishevelled (Dsh), Flamingo (Fmi), Strabismus (Stbm) and Prickle (Pk). Fz and Dsh also function in the Wingless (Wg) or canonical Wnt pathway with Fz being the receptor for Wg or Wnt. Thus, PCP is also referred to as the noncanonical Wnt pathway. In the best understood model system for PCP, the wing epithelium, the seven-pass transmembrane receptor Fz and its cytoplasmic binding partner Dsh localize to the distal apical junctions whereas Stbm and Pk form a complex at the proximal side (Fig. 1). The protocadherin Fmi localizes to both sides and mediates the interaction and intercellular signaling between Fz and Stbm on opposite cell membranes. This asymmetry leads to cellautonomous effects on cell polarity, mainly through activation of small GTPases of the Rho superfamily (Fig. 1), but also to non-autonomous effects with transmission of polarity from cell to cell across the tissue.
Fig. 1 Model for PCP signalling (left) and pupal wing with distorted wing hairs in clones (marked green) (right).
The role of proton transporters in PCP
We have previously identified an unexpected connection between proton transporters and PCP protein localization with the help of a genome-wide RNAi screen in Drosophila S2 cells. The Fz-mediated recruitment of Dsh to the plasma membrane was found to be dependent on pH gradients. The results suggested that the direct interaction between Fz and Dsh is stabilized by a pH and charge-dependent interaction of the basic DEP domain of Dsh with the membrane lipid phosphatidic acid (PA). Recently, we and others found that the V-ATPase subunit, VhaPRR (also called the (pro)renin receptor (PRR) in mammals), is required for Wg and PCP signaling (Hermle et al, 2010; Buechling et al, 2010; Cruciat et al, 2010). These findings raise exciting questions on the role of pH in the establishment of PCP.
The role of pH in regulating cell polarity
As many actin-associated factors function in a pH-dependent manner, proton transport is important for the polarization of the actin cytoskeleton. Protons can also alter the inner surface charge of the plasma membrane. A number of phospholipids such as PA and phosphatidylinositol lipids have phosphate headgroups with pKa values in the physiological range and can, therefore, be protonated at neutral pH. We and others have previously demonstrated that the electrostatic interaction of these lipids with proteins containing basic domains can be controlled by proton pumps and transporters (Simons et al, 2009; Young et al, 2010). These interactions could be particularly important during the polarization of migrating cells. Here, phospholipids are required for the asymmetric membrane recruitment of cytoskeletal regulators and the formation of a leading edge membrane. Our focus is the directional cell migration induced by electric fields (EFs) (also referred to as electrotaxis) where we could recently show that the V-ATPase is needed for electrotaxis of keratinocytes. Currently, we are using mathematical modeling and simulations to understand the interplay of EFs, proton diffusion and cell shape changes essential for directional cell migration.
Fig. 2 Migrating kerotinocyte in the electric field (left) and migrating cells in the Drosophila wing disc (right).
The role of pH and electric fields in wound healing
Electric fields also influence cells and tissues in vivo. Classical studies by Lund, Jaffe, Borgens and others demonstrated that changes in voltage gradients are often correlated with morphogenetic events and patterning across plants and animal kingdoms. It was also convincingly shown that these electric currents were not merely physiological correlates of housekeeping events, but rather provided specific signals regulating cell behaviour during development and regenerative repair. Examples of endogenous currents affecting morphogenetic processes include the polarization of the alga Fucus serratus, the patterning of chick and frog embryos during gastrulation and neurulation and the guidance of a wide range of migratory cell types from vertebrate species. In many of these cases, the findings suggested that the existence of the electric field may precede the movement or differentiation of the cells, thus providing early and spatially instructive signal. This is best understood in epithelial wounds where the planar EF ensures that cells are automatically provided with a vector for directional cell migration and cell division towards the position of the damage. Therefore, one great interest of the lab is to understand the effect of electric fields on cell polarization during wound healing. For this, we are studying wound healing of cultured human keratinocytes but also wound healing of the Drosophila epidermis.
Fig. 3 Schematic representation of electric field (EF) generation in an epithelial wound (left) and electric field distribution around a kerotinocyte migrating in an electric field (right).