Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain
- Department of Neuroscience, University of Virginia, United States
- Department of Biochemistry & Molecular Genetics, University of Virginia, United States
- Department of Cell & Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, United States
- Department of Otolaryngology-Head & Neck Surgery, University of Virginia, United States
- Department of Human Molecular Genetics and Biochemistry, Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Israel
- Genetically Engineered Murine Model (GEMM) Core, University of Virginia, United States
- Department of Cell Biology, University of Virginia, United States
Figures
Figure 1
Noise-induced lesions in the F-actin cores of stereocilia is repaired by actin remodeling.
(A) Cartoon showing cross section of the organ of Corti. (B) Cartoon depicting side view of an inner hair cell (IHC), with a gap in stereocilia F-actin. (C) Representative image of an IHC with a gap …
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Figure 1—source data 1
Quantification of stereocilia gap frequency, hair cell death, and number of stereocilia in control and noise-exposed mice.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig1-data1-v2.xlsx
Figure 2
Xin actin binding repeat containing 2 (XIRP2) is enriched at gaps in stereocilia F-actin cores.
(A) XIRP2 immunostaining is enriched at noise-induced gaps in phalloidin staining in inner hair cells (IHCs) (yellow arrows). (B) XIRP2 immunostaining is enriched at naturally occurring …
Figure 3
The short isoform of Xin actin binding repeat containing 2 (XIRP2) is enriched at gaps and remains there after repair.
(A) Diagram of Xirp2 gene structure and isoforms indicating the positions encoding the epitopes recognized by antibodies used in the figure. (B) XIRP2 enrichment at gaps (yellow arrows) in inner …
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Figure 3—source data 1
Quantification of number of Xin actin binding repeat containing 2 (XIRP2)-enriched sites in stereocilia in control and noise-exposed mice.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig3-data1-v2.xlsx
Figure 4
Xin actin binding repeat containing 2 (XIRP2) is required for the repair of gaps.
(A) Gaps (yellow arrows) in stereocilia F-actin in utricle hair cells from P6 Xirp2 knockout mice. (B) There is a significantly larger percentage of utricle hair cells with gaps in P20 Xirp2 …
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Figure 4—source data 1
Quantification of stereocilia gap frequency, hair cell death, and number of stereocilia in wild-type (WT) and Xirp2 knockout (KO) hair cells.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig4-data1-v2.xlsx
Figure 5
Monomeric γ-actin is no longer recruited to gaps in the absence of Xin actin binding repeat containing 2 (XIRP2).
(A) Representative image of co-labeling of γ-actin and DNAseI enrichment in a noise-induced gap in inner hair cell (IHC) stereocilia F-actin cores. Yellow arrow indicates the site of the gap. (B) …
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Figure 5—source data 1
Quantification of immunofluorescence of γ-actin, β-actin, and espin at stereocilia gaps.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig5-data1-v2.xlsx
Figure 6
The C-terminal domain of Xin actin binding repeat containing 2 (XIRP2) is required for its recruitment to gaps, and its deletion in the Xirp2ΔLIM/CTD mice causes an accumulation of gaps and mild hearing loss.
(A) Diagram of Xirp2 gene structure and isoforms indicating the position of the LIM domain and the region of alternative reading frame targeted in the generation of the Xirp2ΔLIM/CTD mice. (B) …
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Figure 6—source data 1
Quantification of immunofluorescence of Xin actin binding repeat containing 2 (XIRP2), γ-actin at stereocilia gaps, and hair cell numbers and gap frequency in wild-type (WT), Xirp2 knockout (KO), and Xirp2ΔLIM/CTD mice.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig6-data1-v2.xlsx
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Figure 6—source data 2
Auditory brainstem response (ABR) data of wild-type (WT) and Xirp2ΔLIM/CTD mice.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig6-data2-v2.xlsx
Figure 7
The C-terminal domain (CTD) of Xin actin binding repeat containing 2 (XIRP2) recognizes stress fiber strain sites.
(A) Representative image of a bent stereocilium, characterized by local F-actin depolymerization (reduced phalloidin signal) and enrichment of XIRP2. (B) Representative images and cartoon of actin …
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Figure 7—source data 1
Normalized fluorescence intensity fold changes, of FL (full-length), LIM_only, and C-terminal domain (CTD) (C-term domain) of Xin actin binding repeat containing 2 (XIRP2) tagged with EGFP, after laser ablation.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig7-data1-v2.xlsx
Figure 8
Xin actin binding repeat containing 2 (XIRP2) interacts with monomeric and filamentous actin through distinct domains.
(A) Endogenous β- and γ-actin co-immunoprecipitate with heterologously expressed short XIRP2. NIH 3T3 cells were transfected with the EGFP or EGFP-XIRP2 construct as indicated on the top of each …
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Figure 8—source data 1
Western blots showing co-immunoprecipitation (Co-IP) of EGFP-Xin actin binding repeat containing 2 (XIRP2) with β- and γ-actin, Co-IP of EGFP-XIRP2 with wild-type (WT) and polymerization-incompetent γ-actin, and Co-IP of EGFP-tagged full-length and C-terminal domain (CTD) of XIRP2 with polymerization-incompetent γ-actin.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig8-data1-v2.zip
Figure 9
Xirp2 knockout mice are more susceptible to age-related and noise-induced hearing loss.
(A) Auditory brainstem response (ABR) thresholds are significantly elevated in Xirp2 knockout (KO) mice on the C57Bl/6J background at 11–12 weeks of age (***, p<0.001). n=17 wild-type (WT), 9 Xirp2 …
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Figure 9—source data 1
Auditory brainstem response thresholds of aged and noise-exposed Xirp2 knockout (KO) mice on C57Bl/6J or CBA/J background.
- https://cdn.elifesciences.org/articles/72681/elife-72681-fig9-data1-v2.xlsx
Videos
Video 1
EGFP-tagged full-length Xin actin binding repeat containing 2 (XIRP2) is not recruited to stress fiber strain sites.
A focused laser beam was used to induce strain sites in stress fibers of fibroblasts transfected with mApple-actin and EGFP-tagged full-length XIRP2. Cells were imaged for 3 min and 30 s, …
Video 2
EGFP-tagged LIM domain of Xin actin binding repeat containing 2 (XIRP2) is not recruited to stress fiber strain sites.
A focused laser beam was used to induce strain sites in stress fibers of fibroblasts transfected with mApple-actin and EGFP-tagged XIRP2-LIM domain. Cells were imaged for 3 min and 30 s, alternating …
Video 3
EGFP-tagged Xin actin binding repeat containing 2 (XIRP2)-C-terminal domain (CTD) shows rapid recruitment to stress fiber strain sites.
A focused laser beam was used to induce strain sites in stress fibers of fibroblasts transfected with mApple-actin and EGFP-tagged XIRP2-CTD. Cells were imaged for 3 min and 30 s, alternating …
