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

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.

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Gpsm2 and Gαi3 are dynamically expressed at the tips of stereocilia.(a–c) Surface view of whole mounts of rat cochlear sensory epithelium at E17.5 (a) and P7 (b,c) illustrating Gpsm2 (a,b, green) and Gαi3 (c, green) labelling in the actin-rich hair bundle labelled by phalloidin (Ph, magenta). (a) At E17.5, Gpsm2 localizes at the tips of stereocilia at the onset of hair bundle growth (yellow arrows), but also in an asymmetrical crescent in a distal region of the apical membrane of HCs (green asterisks). (b,c) By P7, both proteins accumulate at tips of stereocilia (green), strongly in IHC (yellow arrows) and more weakly in OHC (green arrows). Arrow: inner hair cell (IHC). Bracket: outer hair cell. Scale bars (a–c), 4 μm. (d) Gpsm2 (green) is localized at tips of P8 stereocilia of rat vestibular HC bundles. Ph: phalloidin. Scale bar, 2 μm. (e) At P15, confocal imaging reveals the accumulation of Gαi3 protein at the tip of individual stereocilium. Scale bar 2 μm. (f–h) At P15, STED super-resolution imaging of the Gpsm2-expression domain at an individual stereocilium tip (f). Gpsm2 accumulated at tips of IHC stereocilia (green), above the F-actin labelling (magenta). (f, right panel, g) Acquisition of single plane images in two perpendicular axis as illustrated on the schematic in h reveals the cap-like structure of the Gpsm2 nanodomain. Scale bars, 2 μm. (i) Triple STED labelling reveals two mostly overlapping nanodomains at stereocilia tips with Gpsm2 (green) and Eps8 (magenta) above the F-actin signal (Ph, white). Left images: individual channels for Gpsm2 (top) and Eps8 (middle). The bottom image illustrates the phalloidin channel (grey) with two-colour binary representation of Gpsm2 (green) and Eps8 (magenta), with the overlapping domain (plain white). Scale bar, 2 μm. (j) Isolated long (j) and short (j, inset) stereocilia illustrate the accumulation of the two proteins in the long stereocilium only. Scale bar, 1 μm. (k) Intensity profiles of phalloidin, Gpsm2 and Eps8 from (j) (orange line across tip domain). Immunostainings repeated more than six times.
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f1: Gpsm2 and Gαi3 are dynamically expressed at the tips of stereocilia.(a–c) Surface view of whole mounts of rat cochlear sensory epithelium at E17.5 (a) and P7 (b,c) illustrating Gpsm2 (a,b, green) and Gαi3 (c, green) labelling in the actin-rich hair bundle labelled by phalloidin (Ph, magenta). (a) At E17.5, Gpsm2 localizes at the tips of stereocilia at the onset of hair bundle growth (yellow arrows), but also in an asymmetrical crescent in a distal region of the apical membrane of HCs (green asterisks). (b,c) By P7, both proteins accumulate at tips of stereocilia (green), strongly in IHC (yellow arrows) and more weakly in OHC (green arrows). Arrow: inner hair cell (IHC). Bracket: outer hair cell. Scale bars (a–c), 4 μm. (d) Gpsm2 (green) is localized at tips of P8 stereocilia of rat vestibular HC bundles. Ph: phalloidin. Scale bar, 2 μm. (e) At P15, confocal imaging reveals the accumulation of Gαi3 protein at the tip of individual stereocilium. Scale bar 2 μm. (f–h) At P15, STED super-resolution imaging of the Gpsm2-expression domain at an individual stereocilium tip (f). Gpsm2 accumulated at tips of IHC stereocilia (green), above the F-actin labelling (magenta). (f, right panel, g) Acquisition of single plane images in two perpendicular axis as illustrated on the schematic in h reveals the cap-like structure of the Gpsm2 nanodomain. Scale bars, 2 μm. (i) Triple STED labelling reveals two mostly overlapping nanodomains at stereocilia tips with Gpsm2 (green) and Eps8 (magenta) above the F-actin signal (Ph, white). Left images: individual channels for Gpsm2 (top) and Eps8 (middle). The bottom image illustrates the phalloidin channel (grey) with two-colour binary representation of Gpsm2 (green) and Eps8 (magenta), with the overlapping domain (plain white). Scale bar, 2 μm. (j) Isolated long (j) and short (j, inset) stereocilia illustrate the accumulation of the two proteins in the long stereocilium only. Scale bar, 1 μm. (k) Intensity profiles of phalloidin, Gpsm2 and Eps8 from (j) (orange line across tip domain). Immunostainings repeated more than six times.

Mentions: We evaluated the localization of Gpsm2 and Gαi3 during the development of stereocilia hair bundles using previously characterized specific antibodies6. Gpsm2 was localized at the tip of the nascent hair bundle at embryonic day 17.5 (E17.5), the earliest phase of its formation (Fig. 1a, yellow arrows). Consistent with previous observations, the apical crescent-shaped accumulation of Gpsm2 was also present (Fig. 1a, stars; refs 6, 8). By postnatal day 7 (P7), when stereocilia are rapidly elongating, Gpsm2 and Gαi3 were enriched at the tips of the tallest row of inner hair cell (IHC) stereocilia, the actual sensory receptors receiving 95% of the fibres of the auditory nerve that project to the brain (Fig. 1b,c), but also in vestibular HCs of the ampulla (Fig. 1d). At P15, the enrichment is maintained in the tallest row, whereas we could not detect fluorescence in the middle and small rows (Fig. 1e,f). At this stage, the apical crescent-shaped staining became fragmented or absent, suggesting a gradual loss of these proteins from this zone. Multicolour STimulated Emission Depletion (STED) nanoscopy was used to probe the stereocilia tip compartment and revealed that Gpsm2 was concentrated into a circular cap-like structure (Fig. 1f–h), similar to what was described for myosin 15 (refs 12, 17, 27), above the actin core labelled with phalloidin. Multicolour STED revealed that both Gpsm2 and Eps8 domains mostly overlapped (Fig. 1i). To evaluate the size of the tip domain, we mechanically isolated stereocilia after immunocytochemistry, to obtain perfectly flat structures (Fig. 1j,k). Using fluorescence intensity line-scans along individual long stereocilia labelled with Gpsm2 and Eps8 antibodies and using the full-width at half-maximum (FWHM), we estimated that the tip domain extended ∼200 nm axially at the stereocilia tip (Gpsm2 FWHM=198±59 nm, n=10; Eps8 FWHM=200±63 nm, n=10). These results reveal a narrow stereocilia tip compartment of ∼200 nm where actin filament polymerization is regulated during hair bundle development.


Defective Gpsm2/G α i3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome
Gpsm2 and Gαi3 are dynamically expressed at the tips of stereocilia.(a–c) Surface view of whole mounts of rat cochlear sensory epithelium at E17.5 (a) and P7 (b,c) illustrating Gpsm2 (a,b, green) and Gαi3 (c, green) labelling in the actin-rich hair bundle labelled by phalloidin (Ph, magenta). (a) At E17.5, Gpsm2 localizes at the tips of stereocilia at the onset of hair bundle growth (yellow arrows), but also in an asymmetrical crescent in a distal region of the apical membrane of HCs (green asterisks). (b,c) By P7, both proteins accumulate at tips of stereocilia (green), strongly in IHC (yellow arrows) and more weakly in OHC (green arrows). Arrow: inner hair cell (IHC). Bracket: outer hair cell. Scale bars (a–c), 4 μm. (d) Gpsm2 (green) is localized at tips of P8 stereocilia of rat vestibular HC bundles. Ph: phalloidin. Scale bar, 2 μm. (e) At P15, confocal imaging reveals the accumulation of Gαi3 protein at the tip of individual stereocilium. Scale bar 2 μm. (f–h) At P15, STED super-resolution imaging of the Gpsm2-expression domain at an individual stereocilium tip (f). Gpsm2 accumulated at tips of IHC stereocilia (green), above the F-actin labelling (magenta). (f, right panel, g) Acquisition of single plane images in two perpendicular axis as illustrated on the schematic in h reveals the cap-like structure of the Gpsm2 nanodomain. Scale bars, 2 μm. (i) Triple STED labelling reveals two mostly overlapping nanodomains at stereocilia tips with Gpsm2 (green) and Eps8 (magenta) above the F-actin signal (Ph, white). Left images: individual channels for Gpsm2 (top) and Eps8 (middle). The bottom image illustrates the phalloidin channel (grey) with two-colour binary representation of Gpsm2 (green) and Eps8 (magenta), with the overlapping domain (plain white). Scale bar, 2 μm. (j) Isolated long (j) and short (j, inset) stereocilia illustrate the accumulation of the two proteins in the long stereocilium only. Scale bar, 1 μm. (k) Intensity profiles of phalloidin, Gpsm2 and Eps8 from (j) (orange line across tip domain). Immunostainings repeated more than six times.
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f1: Gpsm2 and Gαi3 are dynamically expressed at the tips of stereocilia.(a–c) Surface view of whole mounts of rat cochlear sensory epithelium at E17.5 (a) and P7 (b,c) illustrating Gpsm2 (a,b, green) and Gαi3 (c, green) labelling in the actin-rich hair bundle labelled by phalloidin (Ph, magenta). (a) At E17.5, Gpsm2 localizes at the tips of stereocilia at the onset of hair bundle growth (yellow arrows), but also in an asymmetrical crescent in a distal region of the apical membrane of HCs (green asterisks). (b,c) By P7, both proteins accumulate at tips of stereocilia (green), strongly in IHC (yellow arrows) and more weakly in OHC (green arrows). Arrow: inner hair cell (IHC). Bracket: outer hair cell. Scale bars (a–c), 4 μm. (d) Gpsm2 (green) is localized at tips of P8 stereocilia of rat vestibular HC bundles. Ph: phalloidin. Scale bar, 2 μm. (e) At P15, confocal imaging reveals the accumulation of Gαi3 protein at the tip of individual stereocilium. Scale bar 2 μm. (f–h) At P15, STED super-resolution imaging of the Gpsm2-expression domain at an individual stereocilium tip (f). Gpsm2 accumulated at tips of IHC stereocilia (green), above the F-actin labelling (magenta). (f, right panel, g) Acquisition of single plane images in two perpendicular axis as illustrated on the schematic in h reveals the cap-like structure of the Gpsm2 nanodomain. Scale bars, 2 μm. (i) Triple STED labelling reveals two mostly overlapping nanodomains at stereocilia tips with Gpsm2 (green) and Eps8 (magenta) above the F-actin signal (Ph, white). Left images: individual channels for Gpsm2 (top) and Eps8 (middle). The bottom image illustrates the phalloidin channel (grey) with two-colour binary representation of Gpsm2 (green) and Eps8 (magenta), with the overlapping domain (plain white). Scale bar, 2 μm. (j) Isolated long (j) and short (j, inset) stereocilia illustrate the accumulation of the two proteins in the long stereocilium only. Scale bar, 1 μm. (k) Intensity profiles of phalloidin, Gpsm2 and Eps8 from (j) (orange line across tip domain). Immunostainings repeated more than six times.
Mentions: We evaluated the localization of Gpsm2 and Gαi3 during the development of stereocilia hair bundles using previously characterized specific antibodies6. Gpsm2 was localized at the tip of the nascent hair bundle at embryonic day 17.5 (E17.5), the earliest phase of its formation (Fig. 1a, yellow arrows). Consistent with previous observations, the apical crescent-shaped accumulation of Gpsm2 was also present (Fig. 1a, stars; refs 6, 8). By postnatal day 7 (P7), when stereocilia are rapidly elongating, Gpsm2 and Gαi3 were enriched at the tips of the tallest row of inner hair cell (IHC) stereocilia, the actual sensory receptors receiving 95% of the fibres of the auditory nerve that project to the brain (Fig. 1b,c), but also in vestibular HCs of the ampulla (Fig. 1d). At P15, the enrichment is maintained in the tallest row, whereas we could not detect fluorescence in the middle and small rows (Fig. 1e,f). At this stage, the apical crescent-shaped staining became fragmented or absent, suggesting a gradual loss of these proteins from this zone. Multicolour STimulated Emission Depletion (STED) nanoscopy was used to probe the stereocilia tip compartment and revealed that Gpsm2 was concentrated into a circular cap-like structure (Fig. 1f–h), similar to what was described for myosin 15 (refs 12, 17, 27), above the actin core labelled with phalloidin. Multicolour STED revealed that both Gpsm2 and Eps8 domains mostly overlapped (Fig. 1i). To evaluate the size of the tip domain, we mechanically isolated stereocilia after immunocytochemistry, to obtain perfectly flat structures (Fig. 1j,k). Using fluorescence intensity line-scans along individual long stereocilia labelled with Gpsm2 and Eps8 antibodies and using the full-width at half-maximum (FWHM), we estimated that the tip domain extended ∼200 nm axially at the stereocilia tip (Gpsm2 FWHM=198±59 nm, n=10; Eps8 FWHM=200±63 nm, n=10). These results reveal a narrow stereocilia tip compartment of ∼200 nm where actin filament polymerization is regulated during hair bundle development.

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.


Related in: MedlinePlus