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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 recycling in the epithelial cells. Schematic model of aPKC transport to the membrane in Rab11-Nuf RE vesicles (left): (1) Vesicles derived from the RE (REdv) containing aPKC move to the apico-lateral site via Rab11-Nuf. (2) aPKC interacts with the apical complexes and becomes active. (3) Active aPKC is able to interact with and phosphorylate Nuf. This phosphorylation release aPKC-Nuf interaction (red dash-line box on the right) (4) inhibiting its apico-lateral localization. aPKC atypical PKC, RE recycling endosome, Nuf nuclear fallout
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Fig5: aPKC recycling in the epithelial cells. Schematic model of aPKC transport to the membrane in Rab11-Nuf RE vesicles (left): (1) Vesicles derived from the RE (REdv) containing aPKC move to the apico-lateral site via Rab11-Nuf. (2) aPKC interacts with the apical complexes and becomes active. (3) Active aPKC is able to interact with and phosphorylate Nuf. This phosphorylation release aPKC-Nuf interaction (red dash-line box on the right) (4) inhibiting its apico-lateral localization. aPKC atypical PKC, RE recycling endosome, Nuf nuclear fallout

Mentions: In contrast, the levels of aPKC depend on aPKC-Nuf interaction. Expression of NufWT or NufS115D do not alter the levels of aPKC, but expression of non-phosphorylatable NufS155A increases aPKC levels in the apico-lateral cortex. This suggests an active transport of aPKC to the apical membrane in Nuf-Rab11 RE. We obtained additional evidence in this direction by interfering with the general RE pathway overexpressing a truncated form of Nuf (NufBRW). We observed a significant increase in the number of Rab11 positive vesicles and the induction of strong phenotypes similar to Rab11-RNAi expression and suggesting a blockage of the recycling pathway. Under this condition, we observed a partial accumulation of aPKC in apical vesicles that are also Rab11 positive, further supporting a Nuf-dependent aPKC recycling mechanism (Additional file 5: Figure S5). However, unlike NufS155A, NufBRW does not accumulate in the membrane, neither aPKC membrane levels are modified, indicating a failure at some level in the fusion of the vesicles, maybe due to the inability of NufBRW to interact with the active aPKC in the membrane. The role of Nuf in the recycling of aPKC is consistent with the cytoplasmic accumulation of aPKC when we block exocytosis in exocyst mutants or RE-vesicle delivery by eliminating Rab11 (Fig. 4), and with the presence of vesicles co-stained with the endocytic endosome GTPase Rab5 and aPKC. Nuf mutant cells show lower levels of apical aPKC suggesting a Nuf-Rab11-RE-mediated transport of aPKC to the apical membrane and aPKC inhibits its own recycling by phosphorylation of Nuf displacing Nuf and Nuf associated-RE from the apico-lateral membrane (Fig. 5).Fig. 5


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 recycling in the epithelial cells. Schematic model of aPKC transport to the membrane in Rab11-Nuf RE vesicles (left): (1) Vesicles derived from the RE (REdv) containing aPKC move to the apico-lateral site via Rab11-Nuf. (2) aPKC interacts with the apical complexes and becomes active. (3) Active aPKC is able to interact with and phosphorylate Nuf. This phosphorylation release aPKC-Nuf interaction (red dash-line box on the right) (4) inhibiting its apico-lateral localization. aPKC atypical PKC, RE recycling endosome, Nuf nuclear fallout
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836198&req=5

Fig5: aPKC recycling in the epithelial cells. Schematic model of aPKC transport to the membrane in Rab11-Nuf RE vesicles (left): (1) Vesicles derived from the RE (REdv) containing aPKC move to the apico-lateral site via Rab11-Nuf. (2) aPKC interacts with the apical complexes and becomes active. (3) Active aPKC is able to interact with and phosphorylate Nuf. This phosphorylation release aPKC-Nuf interaction (red dash-line box on the right) (4) inhibiting its apico-lateral localization. aPKC atypical PKC, RE recycling endosome, Nuf nuclear fallout
Mentions: In contrast, the levels of aPKC depend on aPKC-Nuf interaction. Expression of NufWT or NufS115D do not alter the levels of aPKC, but expression of non-phosphorylatable NufS155A increases aPKC levels in the apico-lateral cortex. This suggests an active transport of aPKC to the apical membrane in Nuf-Rab11 RE. We obtained additional evidence in this direction by interfering with the general RE pathway overexpressing a truncated form of Nuf (NufBRW). We observed a significant increase in the number of Rab11 positive vesicles and the induction of strong phenotypes similar to Rab11-RNAi expression and suggesting a blockage of the recycling pathway. Under this condition, we observed a partial accumulation of aPKC in apical vesicles that are also Rab11 positive, further supporting a Nuf-dependent aPKC recycling mechanism (Additional file 5: Figure S5). However, unlike NufS155A, NufBRW does not accumulate in the membrane, neither aPKC membrane levels are modified, indicating a failure at some level in the fusion of the vesicles, maybe due to the inability of NufBRW to interact with the active aPKC in the membrane. The role of Nuf in the recycling of aPKC is consistent with the cytoplasmic accumulation of aPKC when we block exocytosis in exocyst mutants or RE-vesicle delivery by eliminating Rab11 (Fig. 4), and with the presence of vesicles co-stained with the endocytic endosome GTPase Rab5 and aPKC. Nuf mutant cells show lower levels of apical aPKC suggesting a Nuf-Rab11-RE-mediated transport of aPKC to the apical membrane and aPKC inhibits its own recycling by phosphorylation of Nuf displacing Nuf and Nuf associated-RE from the apico-lateral membrane (Fig. 5).Fig. 5

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