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Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

Cho B, Pierre-Louis G, Sagner A, Eaton S, Axelrod JD - PLoS Genet. (2015)

Bottom Line: This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components.Pk also participates in positive feedback through an unknown mechanism promoting clustering.Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

No MeSH data available.


Related in: MedlinePlus

Amplification of asymmetry in membrane clusters and requirement of Pk for excluding Vang.(A-B”) Clone interface between twin spots expressing either a distal PCP protein fused to ECFP (Fz in A, Dgo in B) or Stbm::EYFP (A’,B’). Note that proximal and distal PCP proteins colocalize in puncta at the clone interface, suggesting that puncta connect PCP complexes of adjacent cells with each other. (C-C”) Clone interface between twin spots expressing either Stbm::ECFP or Stbm::EYFP. (D, E) Quantification of intensity (arbitrary units) along the cell boundaries indicated in A and B, and (F, J) quantification of normalized intensity in C. For F and J, intensity levels of cell boundaries along the clone interface were normalized to the average boundary intensity within the respective twin spot. Clones overexpressing Vang were created adjacent to both wild-type and pk mutant cells by reverse MARCM (see Supporting Information for genotype) (G-I, K-M). (G) Cartoon showing Fz dependent exclusion of Vang-Fmi complexes. (H) Schematic of reverse MARCM clones; red dots indicate vang overexpressing cells (in K-M, red mCherry expressing cells) and yellow dots indicate pk mutant clonal cells facing vang overespressing cells (H, K). (I) Schematic of MARCM experiment. (K) Vang overexpression excludes Vang::YFP from the membrane in neighboring wild-type cells (green arrows), but fails to exclude Vang::YFP in pk mutant cells (yellow dots; yellow arrows indicate membranous Vang::YFP facing vang overexpressing cells in pk mutant cells). (A-C) 16hr APF and (K-M) 26hr APF wing tissues. Scale bars: 5 μm for A-C; 10μm for K-M. Genotypes are (A) y, w, hsflp/+(Y);; ubiP-fz::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (B) y, w, hsflp/+(Y);; ubiP-ECFP::dgo, FRT80 / ubiP-vang::EYFP, FRT80, (C) y, w, hsflp/+(Y);; ubiP-vang::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (K-M) y, w, hsflp/D174GAL4; FRT42D, armP-LacZ /FRT42D, pkpk-sple13, actP-vang::YFP, tubP-GAL80; UAS-mCherry/UAS-vang.
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pgen.1005259.g009: Amplification of asymmetry in membrane clusters and requirement of Pk for excluding Vang.(A-B”) Clone interface between twin spots expressing either a distal PCP protein fused to ECFP (Fz in A, Dgo in B) or Stbm::EYFP (A’,B’). Note that proximal and distal PCP proteins colocalize in puncta at the clone interface, suggesting that puncta connect PCP complexes of adjacent cells with each other. (C-C”) Clone interface between twin spots expressing either Stbm::ECFP or Stbm::EYFP. (D, E) Quantification of intensity (arbitrary units) along the cell boundaries indicated in A and B, and (F, J) quantification of normalized intensity in C. For F and J, intensity levels of cell boundaries along the clone interface were normalized to the average boundary intensity within the respective twin spot. Clones overexpressing Vang were created adjacent to both wild-type and pk mutant cells by reverse MARCM (see Supporting Information for genotype) (G-I, K-M). (G) Cartoon showing Fz dependent exclusion of Vang-Fmi complexes. (H) Schematic of reverse MARCM clones; red dots indicate vang overexpressing cells (in K-M, red mCherry expressing cells) and yellow dots indicate pk mutant clonal cells facing vang overespressing cells (H, K). (I) Schematic of MARCM experiment. (K) Vang overexpression excludes Vang::YFP from the membrane in neighboring wild-type cells (green arrows), but fails to exclude Vang::YFP in pk mutant cells (yellow dots; yellow arrows indicate membranous Vang::YFP facing vang overexpressing cells in pk mutant cells). (A-C) 16hr APF and (K-M) 26hr APF wing tissues. Scale bars: 5 μm for A-C; 10μm for K-M. Genotypes are (A) y, w, hsflp/+(Y);; ubiP-fz::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (B) y, w, hsflp/+(Y);; ubiP-ECFP::dgo, FRT80 / ubiP-vang::EYFP, FRT80, (C) y, w, hsflp/+(Y);; ubiP-vang::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (K-M) y, w, hsflp/D174GAL4; FRT42D, armP-LacZ /FRT42D, pkpk-sple13, actP-vang::YFP, tubP-GAL80; UAS-mCherry/UAS-vang.

Mentions: In contrast to clustering, Pk-dependent Fmi vesicle formation was substantially diminished in fz mutant tissue (Figs 6D and 7D). The requirement of Fz for Pk to stimulate Fmi-Vang-Pk vesicle formation suggests that it is the interaction between full length complexes, most likely within clusters of complexes in opposite orientations that promotes internalization (Fig 9G). This conclusion is further supported by the observation that clones overexpressing Pk, which induce recruitment of Fz to the neighboring cell boundaries and therefore hairs to point toward the clone, simultaneously induce Vang vesicle formation in the adjacent cells (S6 Fig). We note that in fz mutant clones, apicolateral Fmi, the pool from which vesicles may be drawn, is modestly reduced, perhaps by a factor of two [47]. However, there is a greater than 10 fold reduction in vesicle formation, suggesting reduced apicolateral Fmi alone cannot explain the decrease in vesicles. The same is true for vang clones.


Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

Cho B, Pierre-Louis G, Sagner A, Eaton S, Axelrod JD - PLoS Genet. (2015)

Amplification of asymmetry in membrane clusters and requirement of Pk for excluding Vang.(A-B”) Clone interface between twin spots expressing either a distal PCP protein fused to ECFP (Fz in A, Dgo in B) or Stbm::EYFP (A’,B’). Note that proximal and distal PCP proteins colocalize in puncta at the clone interface, suggesting that puncta connect PCP complexes of adjacent cells with each other. (C-C”) Clone interface between twin spots expressing either Stbm::ECFP or Stbm::EYFP. (D, E) Quantification of intensity (arbitrary units) along the cell boundaries indicated in A and B, and (F, J) quantification of normalized intensity in C. For F and J, intensity levels of cell boundaries along the clone interface were normalized to the average boundary intensity within the respective twin spot. Clones overexpressing Vang were created adjacent to both wild-type and pk mutant cells by reverse MARCM (see Supporting Information for genotype) (G-I, K-M). (G) Cartoon showing Fz dependent exclusion of Vang-Fmi complexes. (H) Schematic of reverse MARCM clones; red dots indicate vang overexpressing cells (in K-M, red mCherry expressing cells) and yellow dots indicate pk mutant clonal cells facing vang overespressing cells (H, K). (I) Schematic of MARCM experiment. (K) Vang overexpression excludes Vang::YFP from the membrane in neighboring wild-type cells (green arrows), but fails to exclude Vang::YFP in pk mutant cells (yellow dots; yellow arrows indicate membranous Vang::YFP facing vang overexpressing cells in pk mutant cells). (A-C) 16hr APF and (K-M) 26hr APF wing tissues. Scale bars: 5 μm for A-C; 10μm for K-M. Genotypes are (A) y, w, hsflp/+(Y);; ubiP-fz::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (B) y, w, hsflp/+(Y);; ubiP-ECFP::dgo, FRT80 / ubiP-vang::EYFP, FRT80, (C) y, w, hsflp/+(Y);; ubiP-vang::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (K-M) y, w, hsflp/D174GAL4; FRT42D, armP-LacZ /FRT42D, pkpk-sple13, actP-vang::YFP, tubP-GAL80; UAS-mCherry/UAS-vang.
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pgen.1005259.g009: Amplification of asymmetry in membrane clusters and requirement of Pk for excluding Vang.(A-B”) Clone interface between twin spots expressing either a distal PCP protein fused to ECFP (Fz in A, Dgo in B) or Stbm::EYFP (A’,B’). Note that proximal and distal PCP proteins colocalize in puncta at the clone interface, suggesting that puncta connect PCP complexes of adjacent cells with each other. (C-C”) Clone interface between twin spots expressing either Stbm::ECFP or Stbm::EYFP. (D, E) Quantification of intensity (arbitrary units) along the cell boundaries indicated in A and B, and (F, J) quantification of normalized intensity in C. For F and J, intensity levels of cell boundaries along the clone interface were normalized to the average boundary intensity within the respective twin spot. Clones overexpressing Vang were created adjacent to both wild-type and pk mutant cells by reverse MARCM (see Supporting Information for genotype) (G-I, K-M). (G) Cartoon showing Fz dependent exclusion of Vang-Fmi complexes. (H) Schematic of reverse MARCM clones; red dots indicate vang overexpressing cells (in K-M, red mCherry expressing cells) and yellow dots indicate pk mutant clonal cells facing vang overespressing cells (H, K). (I) Schematic of MARCM experiment. (K) Vang overexpression excludes Vang::YFP from the membrane in neighboring wild-type cells (green arrows), but fails to exclude Vang::YFP in pk mutant cells (yellow dots; yellow arrows indicate membranous Vang::YFP facing vang overexpressing cells in pk mutant cells). (A-C) 16hr APF and (K-M) 26hr APF wing tissues. Scale bars: 5 μm for A-C; 10μm for K-M. Genotypes are (A) y, w, hsflp/+(Y);; ubiP-fz::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (B) y, w, hsflp/+(Y);; ubiP-ECFP::dgo, FRT80 / ubiP-vang::EYFP, FRT80, (C) y, w, hsflp/+(Y);; ubiP-vang::ECFP, FRT80 / ubiP-vang::EYFP, FRT80, (K-M) y, w, hsflp/D174GAL4; FRT42D, armP-LacZ /FRT42D, pkpk-sple13, actP-vang::YFP, tubP-GAL80; UAS-mCherry/UAS-vang.
Mentions: In contrast to clustering, Pk-dependent Fmi vesicle formation was substantially diminished in fz mutant tissue (Figs 6D and 7D). The requirement of Fz for Pk to stimulate Fmi-Vang-Pk vesicle formation suggests that it is the interaction between full length complexes, most likely within clusters of complexes in opposite orientations that promotes internalization (Fig 9G). This conclusion is further supported by the observation that clones overexpressing Pk, which induce recruitment of Fz to the neighboring cell boundaries and therefore hairs to point toward the clone, simultaneously induce Vang vesicle formation in the adjacent cells (S6 Fig). We note that in fz mutant clones, apicolateral Fmi, the pool from which vesicles may be drawn, is modestly reduced, perhaps by a factor of two [47]. However, there is a greater than 10 fold reduction in vesicle formation, suggesting reduced apicolateral Fmi alone cannot explain the decrease in vesicles. The same is true for vang clones.

Bottom Line: This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components.Pk also participates in positive feedback through an unknown mechanism promoting clustering.Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

No MeSH data available.


Related in: MedlinePlus