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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

NufS155A increases aPKC apico-lateral membrane levels. a-b Wing discs of wild-type larvae expressing in the posterior compartment Myc-NufS155A (a) or Myc-NufS155D (b) stained for aPKC (green) or Nuf (anti-Myc, magenta). Upper panels apical views, medial panels sagittal views and lower panels close-up of the above. Arrowheads and arrows point to cortical aPKC in cells located in anterior or posterior compartment of disc. c-f Quantification of aPKC (c-d) and DE-Cad (e-f) levels in epithelia of nuf1/+ heterozygous background wing disc expressing in the posterior Myc-NufS155A (c, e) or Myc-NufS155D (d, f). Fluorescence levels of aPKC or DE-Cad are shown comparing control anterior cells (orange and red) with posterior cells expressing Myc-NufS155A or Myc-NufS155D (blue). Nuf nuclear fallout, aPKC atypical PKC
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Fig3: NufS155A increases aPKC apico-lateral membrane levels. a-b Wing discs of wild-type larvae expressing in the posterior compartment Myc-NufS155A (a) or Myc-NufS155D (b) stained for aPKC (green) or Nuf (anti-Myc, magenta). Upper panels apical views, medial panels sagittal views and lower panels close-up of the above. Arrowheads and arrows point to cortical aPKC in cells located in anterior or posterior compartment of disc. c-f Quantification of aPKC (c-d) and DE-Cad (e-f) levels in epithelia of nuf1/+ heterozygous background wing disc expressing in the posterior Myc-NufS155A (c, e) or Myc-NufS155D (d, f). Fluorescence levels of aPKC or DE-Cad are shown comparing control anterior cells (orange and red) with posterior cells expressing Myc-NufS155A or Myc-NufS155D (blue). Nuf nuclear fallout, aPKC atypical PKC

Mentions: To find if general delivery to the apico-lateral membrane was affected in Nuf mutants, we compared the distribution and levels of DE-Cad (a known RE cargo [21, 22]) and Par-3 markers of adherens junctions, and Crb and aPKC markers of the sub-apical region, in wing discs expressing UAS-Myc-NufS155A or UAS-Myc-NufS155D. While neither of these proteins modified the levels or localization of DE-Cad, Par3 or Crb, expression of the non-phosphorylatable NufS155A induced aPKC membrane accumulation (Fig. 3a). In contrast phosphomimetic NufS155D caused no aPKC accumulation (Fig. 3b). To discard a possible interference of endogenous Nuf protein in the phenotypes, we repeated the experiment in nuf hetero- and homozygous mutants. In both cases the increase in aPKC levels in cells over-expressing NufS155A was maintained (Fig. 3c-d and Additional file 4: Figure S4), but we did not observe any effect in the abundance or localization of the other markers (Fig. 3e-f and Additional file 4: Figure S4). These data indicate that the aPKC-dependent phosphorylation of Nuf exclusively prevents Nuf membrane localization without affecting its binding to the RE machinery and the RE delivery to the apico-lateral membrane and points to aPKC as a cargo of the RE.Fig. 3


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)

NufS155A increases aPKC apico-lateral membrane levels. a-b Wing discs of wild-type larvae expressing in the posterior compartment Myc-NufS155A (a) or Myc-NufS155D (b) stained for aPKC (green) or Nuf (anti-Myc, magenta). Upper panels apical views, medial panels sagittal views and lower panels close-up of the above. Arrowheads and arrows point to cortical aPKC in cells located in anterior or posterior compartment of disc. c-f Quantification of aPKC (c-d) and DE-Cad (e-f) levels in epithelia of nuf1/+ heterozygous background wing disc expressing in the posterior Myc-NufS155A (c, e) or Myc-NufS155D (d, f). Fluorescence levels of aPKC or DE-Cad are shown comparing control anterior cells (orange and red) with posterior cells expressing Myc-NufS155A or Myc-NufS155D (blue). Nuf nuclear fallout, aPKC atypical PKC
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: NufS155A increases aPKC apico-lateral membrane levels. a-b Wing discs of wild-type larvae expressing in the posterior compartment Myc-NufS155A (a) or Myc-NufS155D (b) stained for aPKC (green) or Nuf (anti-Myc, magenta). Upper panels apical views, medial panels sagittal views and lower panels close-up of the above. Arrowheads and arrows point to cortical aPKC in cells located in anterior or posterior compartment of disc. c-f Quantification of aPKC (c-d) and DE-Cad (e-f) levels in epithelia of nuf1/+ heterozygous background wing disc expressing in the posterior Myc-NufS155A (c, e) or Myc-NufS155D (d, f). Fluorescence levels of aPKC or DE-Cad are shown comparing control anterior cells (orange and red) with posterior cells expressing Myc-NufS155A or Myc-NufS155D (blue). Nuf nuclear fallout, aPKC atypical PKC
Mentions: To find if general delivery to the apico-lateral membrane was affected in Nuf mutants, we compared the distribution and levels of DE-Cad (a known RE cargo [21, 22]) and Par-3 markers of adherens junctions, and Crb and aPKC markers of the sub-apical region, in wing discs expressing UAS-Myc-NufS155A or UAS-Myc-NufS155D. While neither of these proteins modified the levels or localization of DE-Cad, Par3 or Crb, expression of the non-phosphorylatable NufS155A induced aPKC membrane accumulation (Fig. 3a). In contrast phosphomimetic NufS155D caused no aPKC accumulation (Fig. 3b). To discard a possible interference of endogenous Nuf protein in the phenotypes, we repeated the experiment in nuf hetero- and homozygous mutants. In both cases the increase in aPKC levels in cells over-expressing NufS155A was maintained (Fig. 3c-d and Additional file 4: Figure S4), but we did not observe any effect in the abundance or localization of the other markers (Fig. 3e-f and Additional file 4: Figure S4). These data indicate that the aPKC-dependent phosphorylation of Nuf exclusively prevents Nuf membrane localization without affecting its binding to the RE machinery and the RE delivery to the apico-lateral membrane and points to aPKC as a cargo of the RE.Fig. 3

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