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GFP fusions of several proteins involved in A-P polarity were created and their localization followed by time-lapse microscopy during polarization in the living C. elegans one-cell embryo. This analysis provides evidence for an establishment phase and a maintenance phase during AP polarization. The authors were also able to establish a temporal and functional hierarchy between the par genes known to control A-P polarity. For instance, analysis of GFP-Par6 localization in par2 embryos revealed that initial localization of Par6 is independent of Par2.
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Cuenca A.A., Schetter A., Aceto D., Kemphues K., Seydoux G. Polarization of the C. elegans zygote proceeds via distinct establishment and maintenance phases. Development. 130:2003;1255-1265 GFP fusions of several proteins involved in A-P polarity were created and their localization followed by time-lapse microscopy during polarization in the living C. elegans one-cell embryo. This analysis provides evidence for an establishment phase and a maintenance phase during AP polarization. The authors were also able to establish a temporal and functional hierarchy between the par genes known to control A-P polarity. For instance, analysis of GFP-Par6 localization in par2 embryos revealed that initial localization of Par6 is independent of Par2.
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Lin D., Edwards A.S., Fawcett J.P., Mbamalu G., Scott J.D., Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat. Cell Biol. 2:2000;540-547.
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Suzuki A., Yamanaka T., Hirose T., Manabe N., Mizuno K., Shimizu M., Akimoto K., Izumi Y., Ohnishi T., Ohno S. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J. Cell Biol. 152:2001;1183-1196.
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Yamanaka T., Horikoshi Y., Suzuki A., Sugiyama Y., Kitamura K., Maniwa R., Nagai Y., Yamashita A., Hirose T., Ishikawa H.et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells. 6:2001;721-731.
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Garrard S.M., Capaldo C.T., Gao L., Rosen M.K., Macara I.G., Tomchick D.R. Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6. EMBO J. 22:2003;1125-1133 This paper describes the crystal structure of a complex between Cdc42 with the mutation Q61L and the GBD of Par6 (which consists of semiCrib and PDZ), providing a coherent molecular basis to understand the biological function of this complex. These structural studies reveal that the way in which Par6 interacts with Cdc42·GTP is unique and different from the mode in which Cdc42·GTP binds WASP or PAK. This is due to the contribution of the Par6's PDZ domain to Cdc42·GTP binding, which stabilizes the interaction with the partial CRIB domain. These studies further indicate that the structural motifs in the PDZ domain that allow its interaction with 'classical' PDZ partners are still available for protein-protein interactions in presence of Cdc42·GTP. Thus, Par6 can simultaneously bind Cdc42 and other PDZ partners, such as Par3. Finally, FRET analysis indicated that binding of Cdc42·GTP induces a conformational alteration in Par6 that may in turn regulate aPKC activity.
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Remarkable study on the ability of yeast cells to polarize in the absence of any spatial cue, and on the central roles of Cdc42 and the actin cytoskeleton in the process. The mechanism should involve a positive feedback loop involving Cdc42 and actin polymerization. The process can be initiated randomly at the plasma membrane at a point where a stochastic rise in the concentration of activated Cdc42 leads to localized formation of actin cables. This results in a higher delivery rate of vesicles to that point, which, by carrying more activated Cdc42, thus reinforce and stabilize the initial asymmetry. The end-result is full polarization of the yeast cell. The authors suggest that in vivo extrinsic and intrinsic polarity signals might use this feedback loop to establish polarity along a defined axis.
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Wedlich-Soldner R., Altschuler S., Wu L., Li R. Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase. Science. 299:2003;1231-1235 Remarkable study on the ability of yeast cells to polarize in the absence of any spatial cue, and on the central roles of Cdc42 and the actin cytoskeleton in the process. The mechanism should involve a positive feedback loop involving Cdc42 and actin polymerization. The process can be initiated randomly at the plasma membrane at a point where a stochastic rise in the concentration of activated Cdc42 leads to localized formation of actin cables. This results in a higher delivery rate of vesicles to that point, which, by carrying more activated Cdc42, thus reinforce and stabilize the initial asymmetry. The end-result is full polarization of the yeast cell. The authors suggest that in vivo extrinsic and intrinsic polarity signals might use this feedback loop to establish polarity along a defined axis.
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The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl
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This paper identifies Lgl as a Par6-aPKC phosphorylation target and proposes a simple model for the accumulation of fate determinants at the basal pole of asymmetrically dividing neuroblasts based on the inhibition of Lgl at the opposite pole. Using immunoprecipitation with anti-Par6 antibody in Drosophila embryos followed by mass spectroscopy sequencing, the authors report the presence of Lgl in the Par6-aPKC complex. The authors show that Lgl is phosphorylated by aPKC and that phosphorylated Lgl is inactive and cytoplasmic. They therefore suggest that phosphorylation of Lgl by the apical Par6-aPKC complex serves to inactivate it at the apical pole of neuroblasts, thus restricting its activity to the basal pole, where it directs the process of localizing Miranda and associated basal determinants.
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Yamanaka T., Horikoshi Y., Sugiyama Y., Ishiyama C., Suzuki A., Hirose T., Iwamatsu A., Shinohara A., Ohno S. Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity. Current Biology. 13:2003;734-743 This study identifies mLgl-1 and mLgl-2 as direct phosphorylation targets of aPKCλ. Initially identified as Par6 interactors, mLgl-1 and mLgl-2 were found in complexes containing aPKCλ and Par6B, but not Par3. mLgl is shown to transiently colocalize with aPKC and Par6 at points of cell-cell contact in polaryzing MDCK cells. As polarity is established, mLgl is segregated from the complex to the baso-lateral domain and this correlates with an increase in its phosphorylation by aPKC.
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These two papers present complementary results that identify a temporal and genetical hierarchy in the way three interacting molecular complexes regulate epithelial apico-basal polarity in Drosophila embryos. A precise temporal observation of mutant phenotypes, alone and in combination, together with a detailed analysis of alterations in protein localization, allowed the authors to show that a major function of the apical Crumbs complex is to restrict the activity of the Scribble complex to a more basal position. In turn, the Scribble complex mainly acts to antagonize the activity of the more apical Baz/Par3-Par6-aPKC complex, which appears to play a more central role in positioning the ZA.
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This paper describes the interaction of Par6 with PALS1 in polarized MDCK cells, thus revealing a molecular link between the two protein complexes that localize to the apical region of these cells. This interaction involves the PDZ domain of Par6 and the N terminus of PALS1 and is functionally important for the correct apico-basal polarization of MDCK cells.
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Cai Y., Yu F., Lin S., Chia W., Yang X. Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions. Cell. 112:2003;51-62 Through the analysis of double mutant combinations, the authors elegantly show that the apical Baz/Par3-Par6-aPKC complex acts redundantly with a second signaling complex, the Pins-Gαi complex, to regulate mitotic spindle positioning in asymmetrically dividing neuroblasts in Drosophila.
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