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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.


Endocytosis of membranous Pk and Vang.Windows were created over the wing of live 24-26h APF pupae by opening the pupal case and cuticle, and then incubated in FM4-64 solution. Apical or sub-apical puncta for GFP::Pk (green in A) and Vang::YFP (green in B) were then visualized. (A) Many GFP::Pk positive cytosolic puncta (GFP::Pk driven by ptc-GAL4) co-labeled with FM4-64. (B) In pk overexpressing clones (outlined), Vang::YFP frequently co-labeled with FM4-64. Double positive puncta for GFP::Pk, or Vang::YFP, and FM4-64 are indicated with arrowheads (see the manuscript for quantification). Scale bars: 10μm. Genotypes are (A) ptc-GAL4/+; UAS-gfp::pk, (B) actP>CD2>GAL4/y, w, hsflp; UAS-pk/+; actP-vang::YFP.
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pgen.1005259.g008: Endocytosis of membranous Pk and Vang.Windows were created over the wing of live 24-26h APF pupae by opening the pupal case and cuticle, and then incubated in FM4-64 solution. Apical or sub-apical puncta for GFP::Pk (green in A) and Vang::YFP (green in B) were then visualized. (A) Many GFP::Pk positive cytosolic puncta (GFP::Pk driven by ptc-GAL4) co-labeled with FM4-64. (B) In pk overexpressing clones (outlined), Vang::YFP frequently co-labeled with FM4-64. Double positive puncta for GFP::Pk, or Vang::YFP, and FM4-64 are indicated with arrowheads (see the manuscript for quantification). Scale bars: 10μm. Genotypes are (A) ptc-GAL4/+; UAS-gfp::pk, (B) actP>CD2>GAL4/y, w, hsflp; UAS-pk/+; actP-vang::YFP.

Mentions: To determine whether the Pk dependent puncta are vesicles resulting from an endocytic process, FM4-64 dye uptake assays were carried out. As FM4-64 cannot penetrate the plasma membrane of live cells, FM4-64 positive puncta indicate endocytic vesicles from internalization of plasma membrane [51]. Live pupae with exposed wings were briefly bathed in FM4-64 and live wings directly subjected to confocal microscopy. To assay for internalization of apical [Fmi-Vang-Pk] complexes, apical and subapical puncta were analyzed. Of GFP::Pk (driven by ptc-GAL4) positive puncta, 36 to 53% (five wings; Fig 8A) were positive for FM4-64. Similarly, when Pk was clonally overexpressed in the presence of Vang::YFP 55 to 74% of Vang::YFP positive puncta were positive for FM4-64 (Fig 8B;five wings tested). This fraction was increased compared to regions not overexpressing Pk (22 to 41%; five wings tested). Note that these percentages are expected to be lower bounds of the true values due to brief exposure to the dye. These results support the idea that Pk mediates endocytosis of Vang. We then asked whether endogenous levels of Vang vesicles depend on endogenous Pk. Consistent with our conclusion, in the absence of Pk (pkpk-sple mutant clones), the number of Vang::YFP positive puncta was reduced to 77% of that in wildtype cells (77± 9%, p = 0.0025; Vang::YFP puncta counted from 412 mutant and 1000 wildtype cells in pkpk-sple mutant clones in five wings). Taken together, these results demonstrate that Vang (together with Fmi) is endocytosed with Pk, and that increased Pk stimulates this internalization. Not surprisingly, these data also indicate that a fraction of Vang::YFP positive vesicles are independent of Pk, perhaps representing distinct recycling and/or biosynthetic pathways.


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)

Endocytosis of membranous Pk and Vang.Windows were created over the wing of live 24-26h APF pupae by opening the pupal case and cuticle, and then incubated in FM4-64 solution. Apical or sub-apical puncta for GFP::Pk (green in A) and Vang::YFP (green in B) were then visualized. (A) Many GFP::Pk positive cytosolic puncta (GFP::Pk driven by ptc-GAL4) co-labeled with FM4-64. (B) In pk overexpressing clones (outlined), Vang::YFP frequently co-labeled with FM4-64. Double positive puncta for GFP::Pk, or Vang::YFP, and FM4-64 are indicated with arrowheads (see the manuscript for quantification). Scale bars: 10μm. Genotypes are (A) ptc-GAL4/+; UAS-gfp::pk, (B) actP>CD2>GAL4/y, w, hsflp; UAS-pk/+; actP-vang::YFP.
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pgen.1005259.g008: Endocytosis of membranous Pk and Vang.Windows were created over the wing of live 24-26h APF pupae by opening the pupal case and cuticle, and then incubated in FM4-64 solution. Apical or sub-apical puncta for GFP::Pk (green in A) and Vang::YFP (green in B) were then visualized. (A) Many GFP::Pk positive cytosolic puncta (GFP::Pk driven by ptc-GAL4) co-labeled with FM4-64. (B) In pk overexpressing clones (outlined), Vang::YFP frequently co-labeled with FM4-64. Double positive puncta for GFP::Pk, or Vang::YFP, and FM4-64 are indicated with arrowheads (see the manuscript for quantification). Scale bars: 10μm. Genotypes are (A) ptc-GAL4/+; UAS-gfp::pk, (B) actP>CD2>GAL4/y, w, hsflp; UAS-pk/+; actP-vang::YFP.
Mentions: To determine whether the Pk dependent puncta are vesicles resulting from an endocytic process, FM4-64 dye uptake assays were carried out. As FM4-64 cannot penetrate the plasma membrane of live cells, FM4-64 positive puncta indicate endocytic vesicles from internalization of plasma membrane [51]. Live pupae with exposed wings were briefly bathed in FM4-64 and live wings directly subjected to confocal microscopy. To assay for internalization of apical [Fmi-Vang-Pk] complexes, apical and subapical puncta were analyzed. Of GFP::Pk (driven by ptc-GAL4) positive puncta, 36 to 53% (five wings; Fig 8A) were positive for FM4-64. Similarly, when Pk was clonally overexpressed in the presence of Vang::YFP 55 to 74% of Vang::YFP positive puncta were positive for FM4-64 (Fig 8B;five wings tested). This fraction was increased compared to regions not overexpressing Pk (22 to 41%; five wings tested). Note that these percentages are expected to be lower bounds of the true values due to brief exposure to the dye. These results support the idea that Pk mediates endocytosis of Vang. We then asked whether endogenous levels of Vang vesicles depend on endogenous Pk. Consistent with our conclusion, in the absence of Pk (pkpk-sple mutant clones), the number of Vang::YFP positive puncta was reduced to 77% of that in wildtype cells (77± 9%, p = 0.0025; Vang::YFP puncta counted from 412 mutant and 1000 wildtype cells in pkpk-sple mutant clones in five wings). Taken together, these results demonstrate that Vang (together with Fmi) is endocytosed with Pk, and that increased Pk stimulates this internalization. Not surprisingly, these data also indicate that a fraction of Vang::YFP positive vesicles are independent of Pk, perhaps representing distinct recycling and/or biosynthetic pathways.

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.