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Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation.

Ren W, Zhang Y, Li M, Wu L, Wang G, Baeg GH, You J, Li Z, Lin X - PLoS Genet. (2015)

Bottom Line: Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions.Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation.Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

No MeSH data available.


Related in: MedlinePlus

Wdp interacts with Dome to promote its internalization for lysosomal degradation.(A-A‴) In wing discs bearing GFP positively marked clones overexpressing Dome-V5 (Act>y+>Gal4, UAS-GFP, UAS-dome-V5), Dome-V5 was mainly localized on the cell membrane (yellow arrows) despite some intracellular punctates (yellow arrowheads). A‴ is the enlarged image of the position labeled by square box in A”. (B-B‴) Coexpression with Wdp alters the subcellular localization of Dome-V5. In wing discs bearing GFP positively marked clones expressing Dome-V5 together with Wdp (Act>y+>Gal4, UAS-GFP, UAS-dome-V5, UAS-wdp), Dome-V5 was depleted from cell membrane but detected as cytoplasmic punctate structures (yellow arrowheads). B‴ is the enlarged image of the position labeled by square box in B”. (C-C‴) The intracellular particles of Dome-V5 in the presence of Wdp were partially colocalized with early endosomes marked by Rab5 staining (white arrows). C‴ is the enlarged image of the position labeled by square box in C”. (D) To detect whether the internalized Dome was degraded, different concentrations of Chloroquine (5, 10 or 20mM) or 10 uM MG132 were used to treat S2 cells transfected with UAS-dome-HA and UAS-wdp. Then cell lysates were analyzed by western blotting with the indicated antibodies. (E) Wdp interacts with Dome in transfected S2 cells. HA-tagged dome, V5-tagged wdp (or no tagged wdp), or pUAST-V5 were transfected individually or together into S2 cells. Cell lysates were immunoprecipitated and analyzed by western blotting with the antibodies indicated. All the wing discs shown here are oriented anterior left, dorsal up. Scale bars, 20μm.
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pgen.1005180.g007: Wdp interacts with Dome to promote its internalization for lysosomal degradation.(A-A‴) In wing discs bearing GFP positively marked clones overexpressing Dome-V5 (Act>y+>Gal4, UAS-GFP, UAS-dome-V5), Dome-V5 was mainly localized on the cell membrane (yellow arrows) despite some intracellular punctates (yellow arrowheads). A‴ is the enlarged image of the position labeled by square box in A”. (B-B‴) Coexpression with Wdp alters the subcellular localization of Dome-V5. In wing discs bearing GFP positively marked clones expressing Dome-V5 together with Wdp (Act>y+>Gal4, UAS-GFP, UAS-dome-V5, UAS-wdp), Dome-V5 was depleted from cell membrane but detected as cytoplasmic punctate structures (yellow arrowheads). B‴ is the enlarged image of the position labeled by square box in B”. (C-C‴) The intracellular particles of Dome-V5 in the presence of Wdp were partially colocalized with early endosomes marked by Rab5 staining (white arrows). C‴ is the enlarged image of the position labeled by square box in C”. (D) To detect whether the internalized Dome was degraded, different concentrations of Chloroquine (5, 10 or 20mM) or 10 uM MG132 were used to treat S2 cells transfected with UAS-dome-HA and UAS-wdp. Then cell lysates were analyzed by western blotting with the indicated antibodies. (E) Wdp interacts with Dome in transfected S2 cells. HA-tagged dome, V5-tagged wdp (or no tagged wdp), or pUAST-V5 were transfected individually or together into S2 cells. Cell lysates were immunoprecipitated and analyzed by western blotting with the antibodies indicated. All the wing discs shown here are oriented anterior left, dorsal up. Scale bars, 20μm.

Mentions: We also examined whether Wdp functions similarly in vivo. We generated flip-out clones overexpressing Dome alone or along with Wdp in the wing and eye imaginal discs. Consistent with previous reports [59–61], Dome-V5, as a transmembrane receptor, was mainly localized on the cell membrane, and also formed some intracellular punctate structures which could correspond to endocytic vesicles (Fig 7A–7A‴). Importantly, coexpression with Wdp caused a significant change in the subcellular localization of Dome (Fig 7B–7B‴). In the presence of Wdp, Dome was totally disappeared from the cell membrane, but was found as intracellular punctates (Fig 7B–7B‴), where they partially colocalized with the early endosome marker Rab5 (Fig 7C–7C‴). In addition, we also observed the same phenomena in the eye discs (S9 Fig). Therefore, these data suggest that enhanced Wdp expression could promote Dome internalization in the wing and eye discs.


Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation.

Ren W, Zhang Y, Li M, Wu L, Wang G, Baeg GH, You J, Li Z, Lin X - PLoS Genet. (2015)

Wdp interacts with Dome to promote its internalization for lysosomal degradation.(A-A‴) In wing discs bearing GFP positively marked clones overexpressing Dome-V5 (Act>y+>Gal4, UAS-GFP, UAS-dome-V5), Dome-V5 was mainly localized on the cell membrane (yellow arrows) despite some intracellular punctates (yellow arrowheads). A‴ is the enlarged image of the position labeled by square box in A”. (B-B‴) Coexpression with Wdp alters the subcellular localization of Dome-V5. In wing discs bearing GFP positively marked clones expressing Dome-V5 together with Wdp (Act>y+>Gal4, UAS-GFP, UAS-dome-V5, UAS-wdp), Dome-V5 was depleted from cell membrane but detected as cytoplasmic punctate structures (yellow arrowheads). B‴ is the enlarged image of the position labeled by square box in B”. (C-C‴) The intracellular particles of Dome-V5 in the presence of Wdp were partially colocalized with early endosomes marked by Rab5 staining (white arrows). C‴ is the enlarged image of the position labeled by square box in C”. (D) To detect whether the internalized Dome was degraded, different concentrations of Chloroquine (5, 10 or 20mM) or 10 uM MG132 were used to treat S2 cells transfected with UAS-dome-HA and UAS-wdp. Then cell lysates were analyzed by western blotting with the indicated antibodies. (E) Wdp interacts with Dome in transfected S2 cells. HA-tagged dome, V5-tagged wdp (or no tagged wdp), or pUAST-V5 were transfected individually or together into S2 cells. Cell lysates were immunoprecipitated and analyzed by western blotting with the antibodies indicated. All the wing discs shown here are oriented anterior left, dorsal up. Scale bars, 20μm.
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Related In: Results  -  Collection

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pgen.1005180.g007: Wdp interacts with Dome to promote its internalization for lysosomal degradation.(A-A‴) In wing discs bearing GFP positively marked clones overexpressing Dome-V5 (Act>y+>Gal4, UAS-GFP, UAS-dome-V5), Dome-V5 was mainly localized on the cell membrane (yellow arrows) despite some intracellular punctates (yellow arrowheads). A‴ is the enlarged image of the position labeled by square box in A”. (B-B‴) Coexpression with Wdp alters the subcellular localization of Dome-V5. In wing discs bearing GFP positively marked clones expressing Dome-V5 together with Wdp (Act>y+>Gal4, UAS-GFP, UAS-dome-V5, UAS-wdp), Dome-V5 was depleted from cell membrane but detected as cytoplasmic punctate structures (yellow arrowheads). B‴ is the enlarged image of the position labeled by square box in B”. (C-C‴) The intracellular particles of Dome-V5 in the presence of Wdp were partially colocalized with early endosomes marked by Rab5 staining (white arrows). C‴ is the enlarged image of the position labeled by square box in C”. (D) To detect whether the internalized Dome was degraded, different concentrations of Chloroquine (5, 10 or 20mM) or 10 uM MG132 were used to treat S2 cells transfected with UAS-dome-HA and UAS-wdp. Then cell lysates were analyzed by western blotting with the indicated antibodies. (E) Wdp interacts with Dome in transfected S2 cells. HA-tagged dome, V5-tagged wdp (or no tagged wdp), or pUAST-V5 were transfected individually or together into S2 cells. Cell lysates were immunoprecipitated and analyzed by western blotting with the antibodies indicated. All the wing discs shown here are oriented anterior left, dorsal up. Scale bars, 20μm.
Mentions: We also examined whether Wdp functions similarly in vivo. We generated flip-out clones overexpressing Dome alone or along with Wdp in the wing and eye imaginal discs. Consistent with previous reports [59–61], Dome-V5, as a transmembrane receptor, was mainly localized on the cell membrane, and also formed some intracellular punctate structures which could correspond to endocytic vesicles (Fig 7A–7A‴). Importantly, coexpression with Wdp caused a significant change in the subcellular localization of Dome (Fig 7B–7B‴). In the presence of Wdp, Dome was totally disappeared from the cell membrane, but was found as intracellular punctates (Fig 7B–7B‴), where they partially colocalized with the early endosome marker Rab5 (Fig 7C–7C‴). In addition, we also observed the same phenomena in the eye discs (S9 Fig). Therefore, these data suggest that enhanced Wdp expression could promote Dome internalization in the wing and eye discs.

Bottom Line: Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions.Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation.Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

No MeSH data available.


Related in: MedlinePlus