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Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis.

Blanco-Sánchez B, Clément A, Fierro J, Washbourne P, Westerfield M - Dis Model Mech (2014)

Bottom Line: We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER).Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis.ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.

ABSTRACT
Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

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Harmonin and Cdh23 bind directly to each other, but not to Ift88. (A) Co-immunoprecipitation. MDCK cells were transfected as indicated by (+) with combinations of DNA vectors encoding Harmonin-HA, mbn-Cdh23-GFP, Cdh23-GFP or IFT88-HA. Input lanes: 1,4,7,10,13. Unbound fraction lanes: 2,5,8,11,14. Bound fraction lanes: 3,6,9,12,15. Immunoprecipitation was performed with an antibody against Harmonin (α-Harm) or GFP (α-GFP). (B,C) Confocal sections of transfected MDCK cells. (B) Double immunolabeling of mbn-Cdh23-GFP (green) and Harmonin (red). (C) Double immunolabeling of mbn-Cdh23-GFP (green) and Ift88 (red). Harmonin-HA: full-length zebrafish Harmonin isoform A fused to HA-tag at the C-terminal. mbn-Cdh23-GFP: zebrafish Cdh23 membrane bound cytoplasmic domain fused to GFP at the C-terminal. Cdh23-GFP: zebrafish Cdh23 cytoplasmic domain fused to GFP at the C-terminal. Ift88-HA: full-length zebrafish Ift88 fused to HA-tag at the C-terminal. Scale bar: 7.5 μm.
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f4-0070547: Harmonin and Cdh23 bind directly to each other, but not to Ift88. (A) Co-immunoprecipitation. MDCK cells were transfected as indicated by (+) with combinations of DNA vectors encoding Harmonin-HA, mbn-Cdh23-GFP, Cdh23-GFP or IFT88-HA. Input lanes: 1,4,7,10,13. Unbound fraction lanes: 2,5,8,11,14. Bound fraction lanes: 3,6,9,12,15. Immunoprecipitation was performed with an antibody against Harmonin (α-Harm) or GFP (α-GFP). (B,C) Confocal sections of transfected MDCK cells. (B) Double immunolabeling of mbn-Cdh23-GFP (green) and Harmonin (red). (C) Double immunolabeling of mbn-Cdh23-GFP (green) and Ift88 (red). Harmonin-HA: full-length zebrafish Harmonin isoform A fused to HA-tag at the C-terminal. mbn-Cdh23-GFP: zebrafish Cdh23 membrane bound cytoplasmic domain fused to GFP at the C-terminal. Cdh23-GFP: zebrafish Cdh23 cytoplasmic domain fused to GFP at the C-terminal. Ift88-HA: full-length zebrafish Ift88 fused to HA-tag at the C-terminal. Scale bar: 7.5 μm.

Mentions: The positive proximity labeling signal for Ift88 with Cdh23, Harmonin and Myo7aa (Fig. 3B,C,F) was unexpected. With the exception of Spag5 (Kersten et al., 2012), previous studies have not reported direct binding between USH proteins and components of the microtubule transport system. Because the proximity assay indicates that proteins are near each other, but not necessarily binding directly, we used co-immunoprecipitation to see whether Ift88 can interact with the USH1 proteins. We expressed HA-tagged Harmonin, HA-tagged Ift88 and GFP-tagged Cdh23 in Madin-Darby canine kidney (MDCK) cells. Protein complexes were immunoprecipitated using anti-Harmonin or anti-GFP antibodies, and protein extracts were analyzed on western blots. Harmonin was able to bind to a membrane-targeted intracellular domain of Cdh23 (mbn-Cdh23; Fig. 4A, lanes 9 and 12), but neither of these USH proteins immunoprecipitated with Ift88 (Fig. 4A, lanes 3, 6, 12 and 15). Consistent with these results, immunolabeling of MDCK cells showed extensive overlap of mbn-Cdh23 with Harmonin (Fig. 4B), but little overlap with Ift88 (Fig. 4C). We obtained essentially the same results when the vectors were transfected into COS7 cells (supplementary material Fig. S10 and data not shown). Interestingly, the intracellular Cdh23 domain, without membrane tethering, did not co-precipitate with Harmonin (Fig. 4A, lane 15), consistent with the interpretation that association of Cdh23 with the membrane is required for USH complex formation. These results show that Ift88 does not co-immunoprecipitate with Cdh23 or Harmonin and suggest that Ift88 might participate in the complex with the three USH1 proteins indirectly.


Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis.

Blanco-Sánchez B, Clément A, Fierro J, Washbourne P, Westerfield M - Dis Model Mech (2014)

Harmonin and Cdh23 bind directly to each other, but not to Ift88. (A) Co-immunoprecipitation. MDCK cells were transfected as indicated by (+) with combinations of DNA vectors encoding Harmonin-HA, mbn-Cdh23-GFP, Cdh23-GFP or IFT88-HA. Input lanes: 1,4,7,10,13. Unbound fraction lanes: 2,5,8,11,14. Bound fraction lanes: 3,6,9,12,15. Immunoprecipitation was performed with an antibody against Harmonin (α-Harm) or GFP (α-GFP). (B,C) Confocal sections of transfected MDCK cells. (B) Double immunolabeling of mbn-Cdh23-GFP (green) and Harmonin (red). (C) Double immunolabeling of mbn-Cdh23-GFP (green) and Ift88 (red). Harmonin-HA: full-length zebrafish Harmonin isoform A fused to HA-tag at the C-terminal. mbn-Cdh23-GFP: zebrafish Cdh23 membrane bound cytoplasmic domain fused to GFP at the C-terminal. Cdh23-GFP: zebrafish Cdh23 cytoplasmic domain fused to GFP at the C-terminal. Ift88-HA: full-length zebrafish Ift88 fused to HA-tag at the C-terminal. Scale bar: 7.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007406&req=5

f4-0070547: Harmonin and Cdh23 bind directly to each other, but not to Ift88. (A) Co-immunoprecipitation. MDCK cells were transfected as indicated by (+) with combinations of DNA vectors encoding Harmonin-HA, mbn-Cdh23-GFP, Cdh23-GFP or IFT88-HA. Input lanes: 1,4,7,10,13. Unbound fraction lanes: 2,5,8,11,14. Bound fraction lanes: 3,6,9,12,15. Immunoprecipitation was performed with an antibody against Harmonin (α-Harm) or GFP (α-GFP). (B,C) Confocal sections of transfected MDCK cells. (B) Double immunolabeling of mbn-Cdh23-GFP (green) and Harmonin (red). (C) Double immunolabeling of mbn-Cdh23-GFP (green) and Ift88 (red). Harmonin-HA: full-length zebrafish Harmonin isoform A fused to HA-tag at the C-terminal. mbn-Cdh23-GFP: zebrafish Cdh23 membrane bound cytoplasmic domain fused to GFP at the C-terminal. Cdh23-GFP: zebrafish Cdh23 cytoplasmic domain fused to GFP at the C-terminal. Ift88-HA: full-length zebrafish Ift88 fused to HA-tag at the C-terminal. Scale bar: 7.5 μm.
Mentions: The positive proximity labeling signal for Ift88 with Cdh23, Harmonin and Myo7aa (Fig. 3B,C,F) was unexpected. With the exception of Spag5 (Kersten et al., 2012), previous studies have not reported direct binding between USH proteins and components of the microtubule transport system. Because the proximity assay indicates that proteins are near each other, but not necessarily binding directly, we used co-immunoprecipitation to see whether Ift88 can interact with the USH1 proteins. We expressed HA-tagged Harmonin, HA-tagged Ift88 and GFP-tagged Cdh23 in Madin-Darby canine kidney (MDCK) cells. Protein complexes were immunoprecipitated using anti-Harmonin or anti-GFP antibodies, and protein extracts were analyzed on western blots. Harmonin was able to bind to a membrane-targeted intracellular domain of Cdh23 (mbn-Cdh23; Fig. 4A, lanes 9 and 12), but neither of these USH proteins immunoprecipitated with Ift88 (Fig. 4A, lanes 3, 6, 12 and 15). Consistent with these results, immunolabeling of MDCK cells showed extensive overlap of mbn-Cdh23 with Harmonin (Fig. 4B), but little overlap with Ift88 (Fig. 4C). We obtained essentially the same results when the vectors were transfected into COS7 cells (supplementary material Fig. S10 and data not shown). Interestingly, the intracellular Cdh23 domain, without membrane tethering, did not co-precipitate with Harmonin (Fig. 4A, lane 15), consistent with the interpretation that association of Cdh23 with the membrane is required for USH complex formation. These results show that Ift88 does not co-immunoprecipitate with Cdh23 or Harmonin and suggest that Ift88 might participate in the complex with the three USH1 proteins indirectly.

Bottom Line: We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER).Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis.ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.

ABSTRACT
Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

Show MeSH
Related in: MedlinePlus