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Defective Gpsm2/G α i3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome

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ABSTRACT

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gαi3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ∼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gαi3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gαi3 in the regulation of actin dynamics in epithelial and neuronal tissues.

No MeSH data available.


Gpsm2 and Gαi3 depend upon myosin 15 to reach stereocilia tips but not for their targeting at HC apical membrane.(a,b) Immunocytochemistry for Gpsm2 (a) or Gαi3 (b) shows both protein localization at the tips of stereocilia (green asterisks), and at the apical membrane of HC as a crescent-shape (yellow dashed-line) from controls of shaker 2 (sh2/+). Homozygous mutation (sh2/sh2) leads to a loss of Gpsm2 and Gαi3 stereocilia tip staining, whereas the apical crescent is maintained. (c) Immunocytochemistry for Gpsm2 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gnai3, but absent from Gnai3 cKO. (d) Reciprocally, immunocytochemistry for Gαi3 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gpsm2, but absent from Gpsm2 cKO. Scale bars, 4 μm. The immunostainings were repeated four times.
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f5: Gpsm2 and Gαi3 depend upon myosin 15 to reach stereocilia tips but not for their targeting at HC apical membrane.(a,b) Immunocytochemistry for Gpsm2 (a) or Gαi3 (b) shows both protein localization at the tips of stereocilia (green asterisks), and at the apical membrane of HC as a crescent-shape (yellow dashed-line) from controls of shaker 2 (sh2/+). Homozygous mutation (sh2/sh2) leads to a loss of Gpsm2 and Gαi3 stereocilia tip staining, whereas the apical crescent is maintained. (c) Immunocytochemistry for Gpsm2 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gnai3, but absent from Gnai3 cKO. (d) Reciprocally, immunocytochemistry for Gαi3 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gpsm2, but absent from Gpsm2 cKO. Scale bars, 4 μm. The immunostainings were repeated four times.

Mentions: Since Gpsm2 and Gnai3 mutants have short stereocilia similar to shaker 2 (sh2; myosin 15 functional ) and whirler (wi) mutants, we hypothesized that these proteins form a larger macromolecular complex. The delivery of whirlin to stereocilia tips requires functional myosin 15, whereas trafficking of myosin 15 can occur independently of whirlin17. We found that Gpsm2 (Fig. 4a,b) and Gαi3 (Fig. 4c,d) were both absent from the stereocilia tips of sh2/sh2 (Fig. 4a,c) and wi/wi HCs at P8 (Fig. 4b,d). These data demonstrate that Gpsm2 and Gαi3 require myosin 15 to be trafficked to the tips of stereocilia. Myosin 15 was still present at the tip of short stereocilia of Gpsm2 and Gnai3 cKOs at P8 (Fig. 4e,f), whereas whirlin localization was absent in Gpsm2 and Gnai3 cKOs (Fig. 4g,h). Importantly, in earlier stages (P4) we observed that the apical crescent of Gpsm2 and Gαi3 was maintained in sh2/sh2 mice (Fig. 5a,b). This result demonstrates that both proteins depend upon different interactions and protein complexes for apical membrane or stereocilia tip traffic within the HC. We also confirmed that the localization of both proteins at the tips of stereocilia was interdependent (Fig. 5c,d) as is the case in many other systems3233. Altogether, our data demonstrate that Gpsm2 and Gαi3 are sorted to the apical membrane and to the stereocilia tip via different protein interactions.


Defective Gpsm2/G α i3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome
Gpsm2 and Gαi3 depend upon myosin 15 to reach stereocilia tips but not for their targeting at HC apical membrane.(a,b) Immunocytochemistry for Gpsm2 (a) or Gαi3 (b) shows both protein localization at the tips of stereocilia (green asterisks), and at the apical membrane of HC as a crescent-shape (yellow dashed-line) from controls of shaker 2 (sh2/+). Homozygous mutation (sh2/sh2) leads to a loss of Gpsm2 and Gαi3 stereocilia tip staining, whereas the apical crescent is maintained. (c) Immunocytochemistry for Gpsm2 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gnai3, but absent from Gnai3 cKO. (d) Reciprocally, immunocytochemistry for Gαi3 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gpsm2, but absent from Gpsm2 cKO. Scale bars, 4 μm. The immunostainings were repeated four times.
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f5: Gpsm2 and Gαi3 depend upon myosin 15 to reach stereocilia tips but not for their targeting at HC apical membrane.(a,b) Immunocytochemistry for Gpsm2 (a) or Gαi3 (b) shows both protein localization at the tips of stereocilia (green asterisks), and at the apical membrane of HC as a crescent-shape (yellow dashed-line) from controls of shaker 2 (sh2/+). Homozygous mutation (sh2/sh2) leads to a loss of Gpsm2 and Gαi3 stereocilia tip staining, whereas the apical crescent is maintained. (c) Immunocytochemistry for Gpsm2 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gnai3, but absent from Gnai3 cKO. (d) Reciprocally, immunocytochemistry for Gαi3 (green) shows protein localization at the tips of stereocilia in IHCs from controls of Gpsm2, but absent from Gpsm2 cKO. Scale bars, 4 μm. The immunostainings were repeated four times.
Mentions: Since Gpsm2 and Gnai3 mutants have short stereocilia similar to shaker 2 (sh2; myosin 15 functional ) and whirler (wi) mutants, we hypothesized that these proteins form a larger macromolecular complex. The delivery of whirlin to stereocilia tips requires functional myosin 15, whereas trafficking of myosin 15 can occur independently of whirlin17. We found that Gpsm2 (Fig. 4a,b) and Gαi3 (Fig. 4c,d) were both absent from the stereocilia tips of sh2/sh2 (Fig. 4a,c) and wi/wi HCs at P8 (Fig. 4b,d). These data demonstrate that Gpsm2 and Gαi3 require myosin 15 to be trafficked to the tips of stereocilia. Myosin 15 was still present at the tip of short stereocilia of Gpsm2 and Gnai3 cKOs at P8 (Fig. 4e,f), whereas whirlin localization was absent in Gpsm2 and Gnai3 cKOs (Fig. 4g,h). Importantly, in earlier stages (P4) we observed that the apical crescent of Gpsm2 and Gαi3 was maintained in sh2/sh2 mice (Fig. 5a,b). This result demonstrates that both proteins depend upon different interactions and protein complexes for apical membrane or stereocilia tip traffic within the HC. We also confirmed that the localization of both proteins at the tips of stereocilia was interdependent (Fig. 5c,d) as is the case in many other systems3233. Altogether, our data demonstrate that Gpsm2 and Gαi3 are sorted to the apical membrane and to the stereocilia tip via different protein interactions.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gαi3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ∼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gαi3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gαi3 in the regulation of actin dynamics in epithelial and neuronal tissues.

No MeSH data available.