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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

Pk is required for post-transcriptional Cul1 complex-mediated core control.Apical patterns of Fz::GFP (green) and Fmi (blue) in pkpk-sple13/pkpk-sple14 pupal wings bearing cul1 knock-down clones (red, RFP) (A). cul1 knock-down clones (red, RFP) were also generated in pkpk-sple14/pksple1 (B) and pkpk-sple14/pkpk30 (C, D) wings. Fmi (green, B-D), PkPk (blue, B) and PKSple (blue, C and D) isoforms were labeled by antibody staining (the anti-Pk antibody recognizes both isoforms; in C’ and D’, signal was assumed to reflect PkSple since the PkPk isoform is not expressed). The presence of either single isoform enables apical core protein enrichment (compare A with B, C, and D) in cul1 knock-down clones. PKSple staining is very weak, but slightly stronger in at 32 h APF compared to 28 h APF (compare C’ with D’). Orientation of PKSple staining corresponds to expected hair direction in pkpk/pkpk-sple mutant wings. D174GAL4 driven GFP::Pk (E), but not HA::pkdC (F), accumulates in cul1 mutant (cul1EX) clones (mutant clones without RFP indicated in Ea and Fa) in 28hr APF wings. In A-D and F, yellow dots are indicating cul1 knock-down or mutant cells. Scale bars: 10μm. Genotypes are (A) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk-sple13; actP>CD2>GAL4, UAS-RFP/armP-fz::EGFP, (B) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pksple1; actP>CD2>GAL4, UAS-RFP/+, (C, D) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk30; actP>CD2>GAL4, UAS-RFP/+, (E) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-GFP::pk/+, (F) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-HA::pkdC/+.
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pgen.1005259.g003: Pk is required for post-transcriptional Cul1 complex-mediated core control.Apical patterns of Fz::GFP (green) and Fmi (blue) in pkpk-sple13/pkpk-sple14 pupal wings bearing cul1 knock-down clones (red, RFP) (A). cul1 knock-down clones (red, RFP) were also generated in pkpk-sple14/pksple1 (B) and pkpk-sple14/pkpk30 (C, D) wings. Fmi (green, B-D), PkPk (blue, B) and PKSple (blue, C and D) isoforms were labeled by antibody staining (the anti-Pk antibody recognizes both isoforms; in C’ and D’, signal was assumed to reflect PkSple since the PkPk isoform is not expressed). The presence of either single isoform enables apical core protein enrichment (compare A with B, C, and D) in cul1 knock-down clones. PKSple staining is very weak, but slightly stronger in at 32 h APF compared to 28 h APF (compare C’ with D’). Orientation of PKSple staining corresponds to expected hair direction in pkpk/pkpk-sple mutant wings. D174GAL4 driven GFP::Pk (E), but not HA::pkdC (F), accumulates in cul1 mutant (cul1EX) clones (mutant clones without RFP indicated in Ea and Fa) in 28hr APF wings. In A-D and F, yellow dots are indicating cul1 knock-down or mutant cells. Scale bars: 10μm. Genotypes are (A) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk-sple13; actP>CD2>GAL4, UAS-RFP/armP-fz::EGFP, (B) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pksple1; actP>CD2>GAL4, UAS-RFP/+, (C, D) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk30; actP>CD2>GAL4, UAS-RFP/+, (E) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-GFP::pk/+, (F) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-HA::pkdC/+.

Mentions: If Pk is a principle target of Cul1 in PCP signaling, the effects of cul1 knock-down or mutation should be suppressed by loss of Pk. To test this, cul1 RNAi clones were generated in pkpk-sple mutant wings and patterns of core proteins were analyzed (Fig 3A). Effects of cul1 RNAi on core protein accumulation were abolished in pkpk-sple mutant wings, such that, for both Fmi and Fz, signal strength inside and outside of clones was indistinguishable (Fig 3A). As expected, asymmetry of Fmi and Fz was disrupted in pkpk-sple wings. Pk is therefore required for the Cul1 complex to act on the core mechanism, and taken together with the full phenocopy of cul1 knock-down or mutation by Pk overexpression (sufficiency; S1 Table), the complete blockade (necessity) suggests that no other Cul1 targets play a significant role in Cul1’s ability to modify core PCP activity.


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)

Pk is required for post-transcriptional Cul1 complex-mediated core control.Apical patterns of Fz::GFP (green) and Fmi (blue) in pkpk-sple13/pkpk-sple14 pupal wings bearing cul1 knock-down clones (red, RFP) (A). cul1 knock-down clones (red, RFP) were also generated in pkpk-sple14/pksple1 (B) and pkpk-sple14/pkpk30 (C, D) wings. Fmi (green, B-D), PkPk (blue, B) and PKSple (blue, C and D) isoforms were labeled by antibody staining (the anti-Pk antibody recognizes both isoforms; in C’ and D’, signal was assumed to reflect PkSple since the PkPk isoform is not expressed). The presence of either single isoform enables apical core protein enrichment (compare A with B, C, and D) in cul1 knock-down clones. PKSple staining is very weak, but slightly stronger in at 32 h APF compared to 28 h APF (compare C’ with D’). Orientation of PKSple staining corresponds to expected hair direction in pkpk/pkpk-sple mutant wings. D174GAL4 driven GFP::Pk (E), but not HA::pkdC (F), accumulates in cul1 mutant (cul1EX) clones (mutant clones without RFP indicated in Ea and Fa) in 28hr APF wings. In A-D and F, yellow dots are indicating cul1 knock-down or mutant cells. Scale bars: 10μm. Genotypes are (A) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk-sple13; actP>CD2>GAL4, UAS-RFP/armP-fz::EGFP, (B) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pksple1; actP>CD2>GAL4, UAS-RFP/+, (C, D) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk30; actP>CD2>GAL4, UAS-RFP/+, (E) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-GFP::pk/+, (F) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-HA::pkdC/+.
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pgen.1005259.g003: Pk is required for post-transcriptional Cul1 complex-mediated core control.Apical patterns of Fz::GFP (green) and Fmi (blue) in pkpk-sple13/pkpk-sple14 pupal wings bearing cul1 knock-down clones (red, RFP) (A). cul1 knock-down clones (red, RFP) were also generated in pkpk-sple14/pksple1 (B) and pkpk-sple14/pkpk30 (C, D) wings. Fmi (green, B-D), PkPk (blue, B) and PKSple (blue, C and D) isoforms were labeled by antibody staining (the anti-Pk antibody recognizes both isoforms; in C’ and D’, signal was assumed to reflect PkSple since the PkPk isoform is not expressed). The presence of either single isoform enables apical core protein enrichment (compare A with B, C, and D) in cul1 knock-down clones. PKSple staining is very weak, but slightly stronger in at 32 h APF compared to 28 h APF (compare C’ with D’). Orientation of PKSple staining corresponds to expected hair direction in pkpk/pkpk-sple mutant wings. D174GAL4 driven GFP::Pk (E), but not HA::pkdC (F), accumulates in cul1 mutant (cul1EX) clones (mutant clones without RFP indicated in Ea and Fa) in 28hr APF wings. In A-D and F, yellow dots are indicating cul1 knock-down or mutant cells. Scale bars: 10μm. Genotypes are (A) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk-sple13; actP>CD2>GAL4, UAS-RFP/armP-fz::EGFP, (B) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pksple1; actP>CD2>GAL4, UAS-RFP/+, (C, D) y, w, hsflp/+; UAS-cul1IR108558, pkpk-sple14/pkpk30; actP>CD2>GAL4, UAS-RFP/+, (E) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-GFP::pk/+, (F) y, w, hsflp/D174GAL4; FRT42D, cul1EX/FRT42D, ubiP-NLS::mRFP; UAS-HA::pkdC/+.
Mentions: If Pk is a principle target of Cul1 in PCP signaling, the effects of cul1 knock-down or mutation should be suppressed by loss of Pk. To test this, cul1 RNAi clones were generated in pkpk-sple mutant wings and patterns of core proteins were analyzed (Fig 3A). Effects of cul1 RNAi on core protein accumulation were abolished in pkpk-sple mutant wings, such that, for both Fmi and Fz, signal strength inside and outside of clones was indistinguishable (Fig 3A). As expected, asymmetry of Fmi and Fz was disrupted in pkpk-sple wings. Pk is therefore required for the Cul1 complex to act on the core mechanism, and taken together with the full phenocopy of cul1 knock-down or mutation by Pk overexpression (sufficiency; S1 Table), the complete blockade (necessity) suggests that no other Cul1 targets play a significant role in Cul1’s ability to modify core PCP activity.

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