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Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

Calero-Cuenca FJ, Espinosa-Vázquez JM, Reina-Campos M, Díaz-Meco MT, Moscat J, Sotillos S - BMC Biol. (2016)

Bottom Line: Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution.We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached.

View Article: PubMed Central - PubMed

Affiliation: CABD, CSIC/JA/UPO, Campus Universidad Pablo de Olavide, Ctra. De Utrera Km. 1, Seville, 41013, Spain.

ABSTRACT

Background: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.

Results: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.

Conclusions: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

No MeSH data available.


Related in: MedlinePlus

aPKC is recycled via Nuf-RE. a-b Confocal images of wing discs containing sec6 (a) or sec5 (b) clones. In the absence of sec6 (a) aPKC accumulates in the mutant cells (red, right panel). In sec5 clones (b) aPKC (blue, right panel) and Nuf (red, middle panel) accumulate. c When Rab11 is silenced, aPKC (red) is disrupted from the apico-lateral cortex (arrows) accumulating in the cytoplasm (right). d Wing disc stained with aPKC (green) and the early recycling endosome marker Rab5 (red). Arrows point to co-stained endosomes. e-f aPKC (red) is reduced in nufKG00314 (e) or nuf1 (f) mutant clones. g-h Fluorescence levels of aPKC comparing wild-type (red) with clone cells (blue) for nufKG00314 (g, left) or nuf1 (h, right). Clones are marked by the absence of GFP (a and b) or β-gal (e-f) or expression of GFP (c) in green. (a-c, e-f) Lower panels show sagittal views of the clones and are marked by arrows. aPKC atypical PKC, RE recycling endosome, GFP green fluorescent protein
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Fig4: aPKC is recycled via Nuf-RE. a-b Confocal images of wing discs containing sec6 (a) or sec5 (b) clones. In the absence of sec6 (a) aPKC accumulates in the mutant cells (red, right panel). In sec5 clones (b) aPKC (blue, right panel) and Nuf (red, middle panel) accumulate. c When Rab11 is silenced, aPKC (red) is disrupted from the apico-lateral cortex (arrows) accumulating in the cytoplasm (right). d Wing disc stained with aPKC (green) and the early recycling endosome marker Rab5 (red). Arrows point to co-stained endosomes. e-f aPKC (red) is reduced in nufKG00314 (e) or nuf1 (f) mutant clones. g-h Fluorescence levels of aPKC comparing wild-type (red) with clone cells (blue) for nufKG00314 (g, left) or nuf1 (h, right). Clones are marked by the absence of GFP (a and b) or β-gal (e-f) or expression of GFP (c) in green. (a-c, e-f) Lower panels show sagittal views of the clones and are marked by arrows. aPKC atypical PKC, RE recycling endosome, GFP green fluorescent protein

Mentions: If aPKC transport to the apical membrane depends on RE, blocking exocytosis in mutants for sec6 or sec5 should increase cytosolic aPKC. Although sec6 mutant cells have very low viability, the few clones recovered accumulated aPKC and Nuf (Fig. 4a, red) and other recycling cargoes, such as Delta [25] and not shown). We obtained similar results with sec5 (Fig. 4b, blue). Accordingly, when eliminating Rab11 with a specific RNAi we observed aPKC was lost from the cell junctions and accumulated sub-apically inside the cell (Fig. 4c). Moreover, we found vesicles co-stained with aPKC and Rab5, an endosomes marker, supporting an active endocytosis of aPKC and reinforcing the idea of aPKC recycling (Fig. 4d).Fig. 4


Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

Calero-Cuenca FJ, Espinosa-Vázquez JM, Reina-Campos M, Díaz-Meco MT, Moscat J, Sotillos S - BMC Biol. (2016)

aPKC is recycled via Nuf-RE. a-b Confocal images of wing discs containing sec6 (a) or sec5 (b) clones. In the absence of sec6 (a) aPKC accumulates in the mutant cells (red, right panel). In sec5 clones (b) aPKC (blue, right panel) and Nuf (red, middle panel) accumulate. c When Rab11 is silenced, aPKC (red) is disrupted from the apico-lateral cortex (arrows) accumulating in the cytoplasm (right). d Wing disc stained with aPKC (green) and the early recycling endosome marker Rab5 (red). Arrows point to co-stained endosomes. e-f aPKC (red) is reduced in nufKG00314 (e) or nuf1 (f) mutant clones. g-h Fluorescence levels of aPKC comparing wild-type (red) with clone cells (blue) for nufKG00314 (g, left) or nuf1 (h, right). Clones are marked by the absence of GFP (a and b) or β-gal (e-f) or expression of GFP (c) in green. (a-c, e-f) Lower panels show sagittal views of the clones and are marked by arrows. aPKC atypical PKC, RE recycling endosome, GFP green fluorescent protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: aPKC is recycled via Nuf-RE. a-b Confocal images of wing discs containing sec6 (a) or sec5 (b) clones. In the absence of sec6 (a) aPKC accumulates in the mutant cells (red, right panel). In sec5 clones (b) aPKC (blue, right panel) and Nuf (red, middle panel) accumulate. c When Rab11 is silenced, aPKC (red) is disrupted from the apico-lateral cortex (arrows) accumulating in the cytoplasm (right). d Wing disc stained with aPKC (green) and the early recycling endosome marker Rab5 (red). Arrows point to co-stained endosomes. e-f aPKC (red) is reduced in nufKG00314 (e) or nuf1 (f) mutant clones. g-h Fluorescence levels of aPKC comparing wild-type (red) with clone cells (blue) for nufKG00314 (g, left) or nuf1 (h, right). Clones are marked by the absence of GFP (a and b) or β-gal (e-f) or expression of GFP (c) in green. (a-c, e-f) Lower panels show sagittal views of the clones and are marked by arrows. aPKC atypical PKC, RE recycling endosome, GFP green fluorescent protein
Mentions: If aPKC transport to the apical membrane depends on RE, blocking exocytosis in mutants for sec6 or sec5 should increase cytosolic aPKC. Although sec6 mutant cells have very low viability, the few clones recovered accumulated aPKC and Nuf (Fig. 4a, red) and other recycling cargoes, such as Delta [25] and not shown). We obtained similar results with sec5 (Fig. 4b, blue). Accordingly, when eliminating Rab11 with a specific RNAi we observed aPKC was lost from the cell junctions and accumulated sub-apically inside the cell (Fig. 4c). Moreover, we found vesicles co-stained with aPKC and Rab5, an endosomes marker, supporting an active endocytosis of aPKC and reinforcing the idea of aPKC recycling (Fig. 4d).Fig. 4

Bottom Line: Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution.We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached.

View Article: PubMed Central - PubMed

Affiliation: CABD, CSIC/JA/UPO, Campus Universidad Pablo de Olavide, Ctra. De Utrera Km. 1, Seville, 41013, Spain.

ABSTRACT

Background: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.

Results: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.

Conclusions: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

No MeSH data available.


Related in: MedlinePlus