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ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus

Expression of ANKS3 in kidney of control mice.(A) ANKS3 expression revealed by Alexa Fluor 488 in green (left column) in a representative cilium stained with acetylated α-tubulin revealed by Alexa Fluor 594 in red (right column); scale bar: 3.5μm. (B) Representative photographs of 6 adult kidneys. ANKS3 staining (revealed by Alexa Fluor 594 in green, left column) co-localises (right column and merge staining in central column) with WGA lectin conjugated to TRITC in red, a marker of podocyte in glomeruli, (scale bar: 90μm) and in tubules stained with Megalin (proximal tubule marker), Tamm-Horsfall protein (Thick ascending limb of Henle’s loop marker) and with AQP2 (collecting duct marker); scale bar: 300μm.
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pone.0136781.g002: Expression of ANKS3 in kidney of control mice.(A) ANKS3 expression revealed by Alexa Fluor 488 in green (left column) in a representative cilium stained with acetylated α-tubulin revealed by Alexa Fluor 594 in red (right column); scale bar: 3.5μm. (B) Representative photographs of 6 adult kidneys. ANKS3 staining (revealed by Alexa Fluor 594 in green, left column) co-localises (right column and merge staining in central column) with WGA lectin conjugated to TRITC in red, a marker of podocyte in glomeruli, (scale bar: 90μm) and in tubules stained with Megalin (proximal tubule marker), Tamm-Horsfall protein (Thick ascending limb of Henle’s loop marker) and with AQP2 (collecting duct marker); scale bar: 300μm.

Mentions: We initially investigated the cellular location of ANKS3 by immunohistochemistry on mouse renal sections. The protein encoded by Anks3was observed in tubular cilia where it co-localises with acetylated α-tubulin (Fig 2A). ANKS3 staining was observed on the whole length of cilia, with a punctuated expression pattern. ANKS3 was also present in glomeruli stained with WGA and at the apical side of tubules (Fig 2B). Double labelling experiments with megalin, Tamm Horsfall protein (TH) and aquaporin 2 (AQP2) showed that ANKS3 is present in the proximal tubules, the thick ascending limb of Henle's loop (TAL) and in the collecting ducts.


ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Expression of ANKS3 in kidney of control mice.(A) ANKS3 expression revealed by Alexa Fluor 488 in green (left column) in a representative cilium stained with acetylated α-tubulin revealed by Alexa Fluor 594 in red (right column); scale bar: 3.5μm. (B) Representative photographs of 6 adult kidneys. ANKS3 staining (revealed by Alexa Fluor 594 in green, left column) co-localises (right column and merge staining in central column) with WGA lectin conjugated to TRITC in red, a marker of podocyte in glomeruli, (scale bar: 90μm) and in tubules stained with Megalin (proximal tubule marker), Tamm-Horsfall protein (Thick ascending limb of Henle’s loop marker) and with AQP2 (collecting duct marker); scale bar: 300μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556665&req=5

pone.0136781.g002: Expression of ANKS3 in kidney of control mice.(A) ANKS3 expression revealed by Alexa Fluor 488 in green (left column) in a representative cilium stained with acetylated α-tubulin revealed by Alexa Fluor 594 in red (right column); scale bar: 3.5μm. (B) Representative photographs of 6 adult kidneys. ANKS3 staining (revealed by Alexa Fluor 594 in green, left column) co-localises (right column and merge staining in central column) with WGA lectin conjugated to TRITC in red, a marker of podocyte in glomeruli, (scale bar: 90μm) and in tubules stained with Megalin (proximal tubule marker), Tamm-Horsfall protein (Thick ascending limb of Henle’s loop marker) and with AQP2 (collecting duct marker); scale bar: 300μm.
Mentions: We initially investigated the cellular location of ANKS3 by immunohistochemistry on mouse renal sections. The protein encoded by Anks3was observed in tubular cilia where it co-localises with acetylated α-tubulin (Fig 2A). ANKS3 staining was observed on the whole length of cilia, with a punctuated expression pattern. ANKS3 was also present in glomeruli stained with WGA and at the apical side of tubules (Fig 2B). Double labelling experiments with megalin, Tamm Horsfall protein (TH) and aquaporin 2 (AQP2) showed that ANKS3 is present in the proximal tubules, the thick ascending limb of Henle's loop (TAL) and in the collecting ducts.

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus