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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

Nuf subcellular distribution is modified by aPKC phosphorylation. a-b Third instar imaginal wing disc (a) and a section is shown in (b). c-d Representation of an apical (1) and a transversal view (2) showing in d the position of apical-lateral markers. e aPKC clones in wing discs (marked by the absence of aPKC, green) accumulate Rab11 (e’ and red in e) and Nuf (e” and blue in e) apically. f-h Subcellular distribution of wild-type (f), non-phosphorylatable (g) and phosphomimetic (h) Nuf in wing discs. Nuf driven by hh-Gal4 accumulates apically. NufS155A (g, magenta) reaches the apico-lateral membrane, marked by aPKC (green, co-localization in white). NufS155D is excluded from the apico-lateral membrane (h). In (f-h), upper panels show apical views, medial panels show sagittal views and lower panels are close-ups of the above. Yellow asterisks mark the apical distribution of the respective Myc-Nuf versions. Scale bar 10 μm. i Quantification of myc (red) compared to DPATJ (blue) levels in epithelia of nuf1 homozygous wing disc expressing Myc-NufWT, Myc-NufS155A or Myc-NufS155D. Picks of DPATJ mark cell membranes. j Apico-cortical accumulation of NufS155A (red) is lost in aPKC-depleted cells marked by the absence of GFP (green, delimited by the yellow dotted line). Par3 (green) was used as membrane marker. k Quantification of Myc (red) and GFP/Par3 (blue) at the boundary between two clone cells (1) or two wild-type cells (2). Nuf nuclear fallout, aPKC atypical PKC, GFP green fluorescent protein.
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Fig2: Nuf subcellular distribution is modified by aPKC phosphorylation. a-b Third instar imaginal wing disc (a) and a section is shown in (b). c-d Representation of an apical (1) and a transversal view (2) showing in d the position of apical-lateral markers. e aPKC clones in wing discs (marked by the absence of aPKC, green) accumulate Rab11 (e’ and red in e) and Nuf (e” and blue in e) apically. f-h Subcellular distribution of wild-type (f), non-phosphorylatable (g) and phosphomimetic (h) Nuf in wing discs. Nuf driven by hh-Gal4 accumulates apically. NufS155A (g, magenta) reaches the apico-lateral membrane, marked by aPKC (green, co-localization in white). NufS155D is excluded from the apico-lateral membrane (h). In (f-h), upper panels show apical views, medial panels show sagittal views and lower panels are close-ups of the above. Yellow asterisks mark the apical distribution of the respective Myc-Nuf versions. Scale bar 10 μm. i Quantification of myc (red) compared to DPATJ (blue) levels in epithelia of nuf1 homozygous wing disc expressing Myc-NufWT, Myc-NufS155A or Myc-NufS155D. Picks of DPATJ mark cell membranes. j Apico-cortical accumulation of NufS155A (red) is lost in aPKC-depleted cells marked by the absence of GFP (green, delimited by the yellow dotted line). Par3 (green) was used as membrane marker. k Quantification of Myc (red) and GFP/Par3 (blue) at the boundary between two clone cells (1) or two wild-type cells (2). Nuf nuclear fallout, aPKC atypical PKC, GFP green fluorescent protein.

Mentions: Nuf is an adaptor of Rab11 to the dynein and kinesin microtubule motor complexes affecting RE distribution [8, 14]. To find out if aPKC regulated Nuf and Rab11 subcellular distribution we studied the imaginal wing disc pseudo-stratified epithelium (Fig. 2a-d). Nuf is found in a uniform punctate distribution in the wild-type disc (Fig. 2e). However, in mutant aPKC clones Nuf accumulates in the subapical region (Fig. 2e”). This accumulation is accompanied by Rab11 accumulation (Fig. 2e’).Fig. 2


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)

Nuf subcellular distribution is modified by aPKC phosphorylation. a-b Third instar imaginal wing disc (a) and a section is shown in (b). c-d Representation of an apical (1) and a transversal view (2) showing in d the position of apical-lateral markers. e aPKC clones in wing discs (marked by the absence of aPKC, green) accumulate Rab11 (e’ and red in e) and Nuf (e” and blue in e) apically. f-h Subcellular distribution of wild-type (f), non-phosphorylatable (g) and phosphomimetic (h) Nuf in wing discs. Nuf driven by hh-Gal4 accumulates apically. NufS155A (g, magenta) reaches the apico-lateral membrane, marked by aPKC (green, co-localization in white). NufS155D is excluded from the apico-lateral membrane (h). In (f-h), upper panels show apical views, medial panels show sagittal views and lower panels are close-ups of the above. Yellow asterisks mark the apical distribution of the respective Myc-Nuf versions. Scale bar 10 μm. i Quantification of myc (red) compared to DPATJ (blue) levels in epithelia of nuf1 homozygous wing disc expressing Myc-NufWT, Myc-NufS155A or Myc-NufS155D. Picks of DPATJ mark cell membranes. j Apico-cortical accumulation of NufS155A (red) is lost in aPKC-depleted cells marked by the absence of GFP (green, delimited by the yellow dotted line). Par3 (green) was used as membrane marker. k Quantification of Myc (red) and GFP/Par3 (blue) at the boundary between two clone cells (1) or two wild-type cells (2). Nuf nuclear fallout, aPKC atypical PKC, GFP green fluorescent protein.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Nuf subcellular distribution is modified by aPKC phosphorylation. a-b Third instar imaginal wing disc (a) and a section is shown in (b). c-d Representation of an apical (1) and a transversal view (2) showing in d the position of apical-lateral markers. e aPKC clones in wing discs (marked by the absence of aPKC, green) accumulate Rab11 (e’ and red in e) and Nuf (e” and blue in e) apically. f-h Subcellular distribution of wild-type (f), non-phosphorylatable (g) and phosphomimetic (h) Nuf in wing discs. Nuf driven by hh-Gal4 accumulates apically. NufS155A (g, magenta) reaches the apico-lateral membrane, marked by aPKC (green, co-localization in white). NufS155D is excluded from the apico-lateral membrane (h). In (f-h), upper panels show apical views, medial panels show sagittal views and lower panels are close-ups of the above. Yellow asterisks mark the apical distribution of the respective Myc-Nuf versions. Scale bar 10 μm. i Quantification of myc (red) compared to DPATJ (blue) levels in epithelia of nuf1 homozygous wing disc expressing Myc-NufWT, Myc-NufS155A or Myc-NufS155D. Picks of DPATJ mark cell membranes. j Apico-cortical accumulation of NufS155A (red) is lost in aPKC-depleted cells marked by the absence of GFP (green, delimited by the yellow dotted line). Par3 (green) was used as membrane marker. k Quantification of Myc (red) and GFP/Par3 (blue) at the boundary between two clone cells (1) or two wild-type cells (2). Nuf nuclear fallout, aPKC atypical PKC, GFP green fluorescent protein.
Mentions: Nuf is an adaptor of Rab11 to the dynein and kinesin microtubule motor complexes affecting RE distribution [8, 14]. To find out if aPKC regulated Nuf and Rab11 subcellular distribution we studied the imaginal wing disc pseudo-stratified epithelium (Fig. 2a-d). Nuf is found in a uniform punctate distribution in the wild-type disc (Fig. 2e). However, in mutant aPKC clones Nuf accumulates in the subapical region (Fig. 2e”). This accumulation is accompanied by Rab11 accumulation (Fig. 2e’).Fig. 2

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