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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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Zebrafish cdh23, ush1c, myo7aa and ift88 mutants have mechanoreceptor structural defects. (A–E) Confocal projections of anterior maculae labeled with phalloidin in (A) wild-type (WT) sibling, (B) cdh23tj264a, (C) ush1cfh293, (D) ift88tz288b and (E) myo7aaty220d mutants. (F–H) Confocal optical sections of double immunolabeling of (F) Harmonin (green) and Ift88 (red), (G) Harmonin (green) and Cdh23 (red), and (H) Harmonin (green) and Myo7aa (red). Individual hair cells are shown. (I) Quantification of total number of hair bundles. n≥6 analyzed anterior maculae. (J) Quantification of total number of hair cells. n≥5 analyzed anterior maculae. (K) Quantification of hair cell death in mutants and wild-type siblings. Number: average number of TUNEL-positive hair cells per macula. n≥16 analyzed anterior maculae. Student’s t-test: **P<0.01, *P<0.05. NS, non significant. Black lines represent ± s.e.m. Scale bars: 5 μm.
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f1-0070547: Zebrafish cdh23, ush1c, myo7aa and ift88 mutants have mechanoreceptor structural defects. (A–E) Confocal projections of anterior maculae labeled with phalloidin in (A) wild-type (WT) sibling, (B) cdh23tj264a, (C) ush1cfh293, (D) ift88tz288b and (E) myo7aaty220d mutants. (F–H) Confocal optical sections of double immunolabeling of (F) Harmonin (green) and Ift88 (red), (G) Harmonin (green) and Cdh23 (red), and (H) Harmonin (green) and Myo7aa (red). Individual hair cells are shown. (I) Quantification of total number of hair bundles. n≥6 analyzed anterior maculae. (J) Quantification of total number of hair cells. n≥5 analyzed anterior maculae. (K) Quantification of hair cell death in mutants and wild-type siblings. Number: average number of TUNEL-positive hair cells per macula. n≥16 analyzed anterior maculae. Student’s t-test: **P<0.01, *P<0.05. NS, non significant. Black lines represent ± s.e.m. Scale bars: 5 μm.

Mentions: We examined the phenotypes resulting from mutations in three of the zebrafish USH1 genes, cdh23, ush1c and myo7aa, and in ift88, a gene that encodes an intraflagellar transport protein known to function in the development of inner ear mechanosensory hair cells (Jones et al., 2008; Kindt et al., 2012; Tsujikawa and Malicki, 2004). We previously showed that ush1c mutants have hearing and balance defects (Phillips et al., 2011), and other studies have shown similar phenotypes in myo7aa (Ernest et al., 2000) and cdh23 (Söllner et al., 2004) zebrafish mutants. To characterize the cellular basis of these defects, we examined the number and the structure of hair cells and stereocilia in the anterior macula of the inner ear. All four mutants showed similar significant structural defects in mechanosensory hair cells, including bent and/or splayed stereocilia (Fig. 1B–E), consistent with the known requirements of USH1 proteins (Bonnet and El-Amraoui, 2012) and Ift88 (Jones et al., 2008) during hair bundle morphogenesis. Additionally, fewer hair cells (~65%) formed stereocilia in all four mutants (Fig. 1I) compared with controls, as previously reported for ush1c mutants (Phillips et al., 2011). We also observed an increase in apoptosis of hair cells in cdh23 and ift88 mutants, as well as in ush1c mutants (Fig. 1K), as we showed previously (Phillips et al., 2011). Despite these defects, the epithelial organization (supplementary material Fig. S1) and number of hair cells formed (Fig. 1J) were relatively normal in the mutants, indicating that initial specification of hair cell fates does not depend upon USH1 or Ift88 function.


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)

Zebrafish cdh23, ush1c, myo7aa and ift88 mutants have mechanoreceptor structural defects. (A–E) Confocal projections of anterior maculae labeled with phalloidin in (A) wild-type (WT) sibling, (B) cdh23tj264a, (C) ush1cfh293, (D) ift88tz288b and (E) myo7aaty220d mutants. (F–H) Confocal optical sections of double immunolabeling of (F) Harmonin (green) and Ift88 (red), (G) Harmonin (green) and Cdh23 (red), and (H) Harmonin (green) and Myo7aa (red). Individual hair cells are shown. (I) Quantification of total number of hair bundles. n≥6 analyzed anterior maculae. (J) Quantification of total number of hair cells. n≥5 analyzed anterior maculae. (K) Quantification of hair cell death in mutants and wild-type siblings. Number: average number of TUNEL-positive hair cells per macula. n≥16 analyzed anterior maculae. Student’s t-test: **P<0.01, *P<0.05. NS, non significant. Black lines represent ± s.e.m. Scale bars: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-0070547: Zebrafish cdh23, ush1c, myo7aa and ift88 mutants have mechanoreceptor structural defects. (A–E) Confocal projections of anterior maculae labeled with phalloidin in (A) wild-type (WT) sibling, (B) cdh23tj264a, (C) ush1cfh293, (D) ift88tz288b and (E) myo7aaty220d mutants. (F–H) Confocal optical sections of double immunolabeling of (F) Harmonin (green) and Ift88 (red), (G) Harmonin (green) and Cdh23 (red), and (H) Harmonin (green) and Myo7aa (red). Individual hair cells are shown. (I) Quantification of total number of hair bundles. n≥6 analyzed anterior maculae. (J) Quantification of total number of hair cells. n≥5 analyzed anterior maculae. (K) Quantification of hair cell death in mutants and wild-type siblings. Number: average number of TUNEL-positive hair cells per macula. n≥16 analyzed anterior maculae. Student’s t-test: **P<0.01, *P<0.05. NS, non significant. Black lines represent ± s.e.m. Scale bars: 5 μm.
Mentions: We examined the phenotypes resulting from mutations in three of the zebrafish USH1 genes, cdh23, ush1c and myo7aa, and in ift88, a gene that encodes an intraflagellar transport protein known to function in the development of inner ear mechanosensory hair cells (Jones et al., 2008; Kindt et al., 2012; Tsujikawa and Malicki, 2004). We previously showed that ush1c mutants have hearing and balance defects (Phillips et al., 2011), and other studies have shown similar phenotypes in myo7aa (Ernest et al., 2000) and cdh23 (Söllner et al., 2004) zebrafish mutants. To characterize the cellular basis of these defects, we examined the number and the structure of hair cells and stereocilia in the anterior macula of the inner ear. All four mutants showed similar significant structural defects in mechanosensory hair cells, including bent and/or splayed stereocilia (Fig. 1B–E), consistent with the known requirements of USH1 proteins (Bonnet and El-Amraoui, 2012) and Ift88 (Jones et al., 2008) during hair bundle morphogenesis. Additionally, fewer hair cells (~65%) formed stereocilia in all four mutants (Fig. 1I) compared with controls, as previously reported for ush1c mutants (Phillips et al., 2011). We also observed an increase in apoptosis of hair cells in cdh23 and ift88 mutants, as well as in ush1c mutants (Fig. 1K), as we showed previously (Phillips et al., 2011). Despite these defects, the epithelial organization (supplementary material Fig. S1) and number of hair cells formed (Fig. 1J) were relatively normal in the mutants, indicating that initial specification of hair cell fates does not depend upon USH1 or Ift88 function.

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