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The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

Fang Q, Indzhykulian AA, Mustapha M, Riordan GP, Dolan DF, Friedman TB, Belyantseva IA, Frolenkov GI, Camper SA, Bird JE - Elife (2015)

Bottom Line: We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows.The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core.Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, United States.

ABSTRACT
The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

No MeSH data available.


Related in: MedlinePlus

Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.(A, B) SEM images of IHC (A) and OHC (B) stereocilia bundles in Myo15+/ΔN (left panels) and Myo15ΔN/ΔN (right panels) hair cells. Higher magnification of the second row IHC stereocilia tips are shown (inset). (C, D) Whole cell current responses (top traces) evoked by graded deflections of the stereocilia bundles (bottom traces) in IHCs (C) and OHCs (D) in Myo15+/ΔN (left) and Myo15ΔN/ΔN (right) hair cells. (E, H) Relationship between the peak MET current and stereocilia bundle displacement in IHCs (E) and OHCs (H) from Myo15+/ΔN (open circles) and Myo15ΔN/ΔN (closed circles) cochleae. (F, I) Time constants of MET adaptation in IHCs (F) and OHCs (I) for Myo15ΔN/ΔN and Myo15+/ΔN. Time constants were determined from a single exponential fit of MET responses evoked by the small bundle deflections of ∼150 nm (see black traces in C, D). (G, J) Percent changes of the MET current 10 ms after a stimulation step (extent of adaptation) as a function of stimulus intensity in IHCs (G) and OHCs (J). The same MET records contribute to all averaged data. Data are mean ± SE. Asterisks indicate statistical significance: *, p < 0.01; **, p < 0.001; ***, p < 0.0001 (t-test of independent variables). Holding potential was −90 mV. Age of the cells: P3-4 + 3–5 days in vitro. SEM images were obtained from cultured samples used for MET recordings. Number of cells: n = 10 (IHCs, Myo15+/ΔN), n = 12 (IHCs, Myo15ΔN/ΔN), n = 7 (OHCs, Myo15+/ΔN), n = 9 (OHCs, Myo15ΔN/ΔN).DOI:http://dx.doi.org/10.7554/eLife.08627.012
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fig5: Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.(A, B) SEM images of IHC (A) and OHC (B) stereocilia bundles in Myo15+/ΔN (left panels) and Myo15ΔN/ΔN (right panels) hair cells. Higher magnification of the second row IHC stereocilia tips are shown (inset). (C, D) Whole cell current responses (top traces) evoked by graded deflections of the stereocilia bundles (bottom traces) in IHCs (C) and OHCs (D) in Myo15+/ΔN (left) and Myo15ΔN/ΔN (right) hair cells. (E, H) Relationship between the peak MET current and stereocilia bundle displacement in IHCs (E) and OHCs (H) from Myo15+/ΔN (open circles) and Myo15ΔN/ΔN (closed circles) cochleae. (F, I) Time constants of MET adaptation in IHCs (F) and OHCs (I) for Myo15ΔN/ΔN and Myo15+/ΔN. Time constants were determined from a single exponential fit of MET responses evoked by the small bundle deflections of ∼150 nm (see black traces in C, D). (G, J) Percent changes of the MET current 10 ms after a stimulation step (extent of adaptation) as a function of stimulus intensity in IHCs (G) and OHCs (J). The same MET records contribute to all averaged data. Data are mean ± SE. Asterisks indicate statistical significance: *, p < 0.01; **, p < 0.001; ***, p < 0.0001 (t-test of independent variables). Holding potential was −90 mV. Age of the cells: P3-4 + 3–5 days in vitro. SEM images were obtained from cultured samples used for MET recordings. Number of cells: n = 10 (IHCs, Myo15+/ΔN), n = 12 (IHCs, Myo15ΔN/ΔN), n = 7 (OHCs, Myo15+/ΔN), n = 9 (OHCs, Myo15ΔN/ΔN).DOI:http://dx.doi.org/10.7554/eLife.08627.012

Mentions: To test whether isoform 1 is a critical component of the transduction machinery, MET currents were measured from Myo15ΔN/ΔN hair cells, which have normal staircase morphology and correctly oriented tip-links (Figure 2 and Figure 5A,B). Whole cell MET current responses of young postnatal IHCs and OHCs (P3-4 + 3–5 days in vitro) were evoked by graded stereocilia deflections using a rigid probe (Figure 5C,D). Maximal MET current amplitudes were not statistically different between mutant Myo15ΔN/ΔN (n = 12) and control Myo15+/ΔN IHCs (n = 10) (Figure 5E). However, the responses to small bundle deflections (150–300 nm) were significantly larger in mutant Myo15ΔN/ΔN IHCs, indicating an increased deflection sensitivity of the transduction apparatus in the absence of isoform 1 (Figure 5E). This sensitivity depends on the mechanical stiffness of a theoretical ‘gating spring’ that is connected to the MET channel (Howard and Hudspeth, 1987). A stiffer gating spring would transmit the maximal opening force to the MET channel at a smaller bundle deflection, resulting in earlier saturation of the current-displacement relationship. This was indeed observed in Myo15ΔN/ΔN IHCs (Figure 5E). Our data show that isoform 1 is not essential for MET responses in IHCs but may contribute to the stiffness of this gating spring. We did not observe a similar increase of MET sensitivity in OHCs (Myo15ΔN/ΔN, n = 9; Myo15+/ΔN, n = 7), perhaps due to the MET current degradation already present in Myo15ΔN/ΔN OHCs compared to Myo15+/ΔN controls (Figure 5H).10.7554/eLife.08627.012Figure 5.Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.


The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

Fang Q, Indzhykulian AA, Mustapha M, Riordan GP, Dolan DF, Friedman TB, Belyantseva IA, Frolenkov GI, Camper SA, Bird JE - Elife (2015)

Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.(A, B) SEM images of IHC (A) and OHC (B) stereocilia bundles in Myo15+/ΔN (left panels) and Myo15ΔN/ΔN (right panels) hair cells. Higher magnification of the second row IHC stereocilia tips are shown (inset). (C, D) Whole cell current responses (top traces) evoked by graded deflections of the stereocilia bundles (bottom traces) in IHCs (C) and OHCs (D) in Myo15+/ΔN (left) and Myo15ΔN/ΔN (right) hair cells. (E, H) Relationship between the peak MET current and stereocilia bundle displacement in IHCs (E) and OHCs (H) from Myo15+/ΔN (open circles) and Myo15ΔN/ΔN (closed circles) cochleae. (F, I) Time constants of MET adaptation in IHCs (F) and OHCs (I) for Myo15ΔN/ΔN and Myo15+/ΔN. Time constants were determined from a single exponential fit of MET responses evoked by the small bundle deflections of ∼150 nm (see black traces in C, D). (G, J) Percent changes of the MET current 10 ms after a stimulation step (extent of adaptation) as a function of stimulus intensity in IHCs (G) and OHCs (J). The same MET records contribute to all averaged data. Data are mean ± SE. Asterisks indicate statistical significance: *, p < 0.01; **, p < 0.001; ***, p < 0.0001 (t-test of independent variables). Holding potential was −90 mV. Age of the cells: P3-4 + 3–5 days in vitro. SEM images were obtained from cultured samples used for MET recordings. Number of cells: n = 10 (IHCs, Myo15+/ΔN), n = 12 (IHCs, Myo15ΔN/ΔN), n = 7 (OHCs, Myo15+/ΔN), n = 9 (OHCs, Myo15ΔN/ΔN).DOI:http://dx.doi.org/10.7554/eLife.08627.012
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Related In: Results  -  Collection

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fig5: Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.(A, B) SEM images of IHC (A) and OHC (B) stereocilia bundles in Myo15+/ΔN (left panels) and Myo15ΔN/ΔN (right panels) hair cells. Higher magnification of the second row IHC stereocilia tips are shown (inset). (C, D) Whole cell current responses (top traces) evoked by graded deflections of the stereocilia bundles (bottom traces) in IHCs (C) and OHCs (D) in Myo15+/ΔN (left) and Myo15ΔN/ΔN (right) hair cells. (E, H) Relationship between the peak MET current and stereocilia bundle displacement in IHCs (E) and OHCs (H) from Myo15+/ΔN (open circles) and Myo15ΔN/ΔN (closed circles) cochleae. (F, I) Time constants of MET adaptation in IHCs (F) and OHCs (I) for Myo15ΔN/ΔN and Myo15+/ΔN. Time constants were determined from a single exponential fit of MET responses evoked by the small bundle deflections of ∼150 nm (see black traces in C, D). (G, J) Percent changes of the MET current 10 ms after a stimulation step (extent of adaptation) as a function of stimulus intensity in IHCs (G) and OHCs (J). The same MET records contribute to all averaged data. Data are mean ± SE. Asterisks indicate statistical significance: *, p < 0.01; **, p < 0.001; ***, p < 0.0001 (t-test of independent variables). Holding potential was −90 mV. Age of the cells: P3-4 + 3–5 days in vitro. SEM images were obtained from cultured samples used for MET recordings. Number of cells: n = 10 (IHCs, Myo15+/ΔN), n = 12 (IHCs, Myo15ΔN/ΔN), n = 7 (OHCs, Myo15+/ΔN), n = 9 (OHCs, Myo15ΔN/ΔN).DOI:http://dx.doi.org/10.7554/eLife.08627.012
Mentions: To test whether isoform 1 is a critical component of the transduction machinery, MET currents were measured from Myo15ΔN/ΔN hair cells, which have normal staircase morphology and correctly oriented tip-links (Figure 2 and Figure 5A,B). Whole cell MET current responses of young postnatal IHCs and OHCs (P3-4 + 3–5 days in vitro) were evoked by graded stereocilia deflections using a rigid probe (Figure 5C,D). Maximal MET current amplitudes were not statistically different between mutant Myo15ΔN/ΔN (n = 12) and control Myo15+/ΔN IHCs (n = 10) (Figure 5E). However, the responses to small bundle deflections (150–300 nm) were significantly larger in mutant Myo15ΔN/ΔN IHCs, indicating an increased deflection sensitivity of the transduction apparatus in the absence of isoform 1 (Figure 5E). This sensitivity depends on the mechanical stiffness of a theoretical ‘gating spring’ that is connected to the MET channel (Howard and Hudspeth, 1987). A stiffer gating spring would transmit the maximal opening force to the MET channel at a smaller bundle deflection, resulting in earlier saturation of the current-displacement relationship. This was indeed observed in Myo15ΔN/ΔN IHCs (Figure 5E). Our data show that isoform 1 is not essential for MET responses in IHCs but may contribute to the stiffness of this gating spring. We did not observe a similar increase of MET sensitivity in OHCs (Myo15ΔN/ΔN, n = 9; Myo15+/ΔN, n = 7), perhaps due to the MET current degradation already present in Myo15ΔN/ΔN OHCs compared to Myo15+/ΔN controls (Figure 5H).10.7554/eLife.08627.012Figure 5.Isoform 1 is not required for MET but influences the deflection sensitivity of IHCs.

Bottom Line: We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows.The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core.Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, United States.

ABSTRACT
The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.

No MeSH data available.


Related in: MedlinePlus