Molecular mechanisms specifying epithelial and neuronal polarity : segregation of membrane domains and polarized trafficking

Cell polarity is involved in the processes of differentiation, proliferation and morphogenesis in both unicellular and multicellular organisms. In polarized cells, such as epithelial cells and neurons, it is possible to distinguish a junctional domain (cell-cell and cell-matrix) and an extra-junctional domain (apical domain in epithelial cells and extra-synaptic domain in neurons). In epithelial cells, assembly of tight junctions (TJs) leads to the formation of apical and basolateral plasma membrane domains, while the functional analogous of these junctions in neurons is still unknown. By organizing protein complexes through protein-protein interaction domains, scaffold proteins play an important role in the assembly of TJs and in the organization of the post-synaptic density (PSD) at post-synaptic sites, and here we have investigated the role of the L27 and PDZ domains for proteinprotein interaction of the small scaffold protein LIN7 in Madin-Darby canine kidney (MDCK) cells and in the motor neuronal NSC-34 cell line. In MDCK cells, we have found that the stable expression of a LIN7 mutantlacking the L27 domain (delL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 partner of the L27 domain, but prevented TJlocalization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domain of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7shRNA experiments), and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53del5) or fused to the L27 domain of LIN7 (L27-IRSp53del5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization, and failed to activate Rac1, a member of the Rho GTPase family crucially involved in actin organization and orientation of apico-basal polarity. These data indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells. In NSC-34 cells, a motor neuron-like hybrid cell line, we examined the role of LIN7 in the localization and function of the IRSp53 partner of its PDZ domain. To this purpose we analyzed the effects of transient expression of IRSp53 and LIN7 constructs (expressed individually or together) in NSC-34 cells. We found that overexpression of IRSp53 induced the formation of an exceeding number of filopodia and pseudo-fiopodia (without detectable filamentous actin), while a number of filopodia comparable to untransfected cells and absence of pseudofilopodia were observed when IRSp53 was coexpressed together with LIN7 and when the L27-IRSp53del5 chimera was expressed. Our data indicate that LIN7- IRSp53 association is also involved in the polarization of neuronal cells by regulating the formation of the precursors of dendritic spines. In order to maintain cell polarity, epithelial cells have also to ensure proper delivery and/or retention of apical and basolateral cargo to their respective target location. Moreover, the recycling of proteins from and to different plasma membrane domains must be finely regulated. In this study, we have used glutamate and GABA transporters differentially located to apical or lateral surface as molecular tool to study the sorting mechanisms regulating the polarized distribution of plasma membrane proteins. In MDCK cells, we have investigated the PKC mediated regulation of the apically located EAAC1 glutamate transporter. We found that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of EAAC1 from the apical surface of polarized MDCK cells to intracellular compartments. The PKC-induced internalization of EAAC1 was also dependent on the calcineurin phosphatase activity, and on the endocytic activity of dynamin, a well known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a targetof these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin, and that this activation is required to mediate internalization of apical EAAC1 in MDCK cells. In sharp contrast, we found that PKC induced a relocalization of a chimeric EAAC1 lacking the apical localization signal from the lateral surface to intracellular compartments, but this redistribution was not calcineurin dependent. We also demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral BGT1 GABA transporter and activated PKCα. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a post-treatment with a PKC specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together our data indicate that PKC activity regulates EAAC1 surface density by inducing its internalization and retention to PKCα labeled recycling endosomes common to apical and basolateral proteins, and suggest that a differential endocytotic machinery takes place at the apical and basolateral surfaces in MDCK cells.