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. 2022 Jun;79(6-8):64-74.
doi: 10.1002/cm.21719. Epub 2022 Jul 30.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization

Lili Zheng  1 , Stephen A Adam  1 , Jaime García-Anoveros  2   3 , Brian J Mitchell  1 , James R Bartles  1   3
Affiliations

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization

Lili Zheng et al. Cytoskeleton (Hoboken). 2022 Jun.

Abstract

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Keywords: actin; espin; hair cell; stereocilia.

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Conflict of interest statement

The authors have no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Validation of Espin over‐expression. (a) TaqMan real‐time PCR based copy number assay for wild type (WT) and espin OE (OE). Data is presented as both male (n of 4 WT and 4 OE), female (n of 4 WT and 7OE) and combined. (b) Domain architecture for the 4 main isoforms of the espin gene. (c) Western blot analysis of espin protein levels in the testes (Espin 1, 2 representative animals), in the cerebellum (espin 2, 2 representative animals), and in the kidney (espin 4, 2 representative animals). (d) Normalized quantification of espin levels (n = 6; error bars represent S.D., t‐test *p < 0.05)
FIGURE 2
FIGURE 2
Espin‐OE alters cochlear stereocilia length. (a‐b) Antibody staining of espin (green) together with phalloidin (red) of wild type (a) and espin‐OE (b) cochlea depicting a grossly normal organization of outer hair cells and a mild disorganization of inner hair cells. (c‐g) Top and side projection of wild type (c, e) and espin‐OE (d, f) inner hair cells together with the quantification of tallest row stereocilia length (g, n = 140 stereocilia from ≤8 cochlea per condition). (h‐l) Tilted projection and side projection of wild type (h, J) and espin OE (I, K), together with quantification of tallest row stereocilia length (l, n = 120 stereocilia from ≤9 cochlea per condition). Statistics performed were Students t‐test and *** represents a p value <0.0001. Scale bars are 5 μm
FIGURE 3
FIGURE 3
Espin‐OE alters utricle stereocilia length. (a‐b) Antibody staining of espin (green) together with phalloidin (red) of wild type (a) and espin‐OE (b) utricle hair cells. (c‐d) TEM micrographs of wild type (c) and espin‐OE (d) utricle hair cells. (e‐g) Structured illumination microscopy of isolated utricle stereocilia from wild type (e) and espin‐OE (f) cells. (g) Quantification of stereocilia width measured at both in the middle and at the tip of the isolated stereocilia (n = 105 stereocilia each condition). Scale bar is 5 μm in a‐b and 1 μm in c‐f
FIGURE 4
FIGURE 4
Espin‐OE drives mis‐localization of stereocilia proteins. (a‐f) Antibody staining of P10 cochlea from wild type animals on the left and espin‐OE animals on the right stained with phalloidin (red) and antibodies to GNAI3 (a), GPSM2 (b), EPS8 (c), WHRN (d), MYO15A‐L (e), and MYO15A‐L + S (f) in green. Images are representative of n > 3 animals. Scale bar is 5 μm
FIGURE 5
FIGURE 5
Espin has anti‐capping function. (a) Spectrofluorimetric pyrene‐Actin polymerization assay examining the block of polymerization with the addition of capping protein (CP) and an increasing anti‐capping effect with increasing amounts (μg) of wild type espin2B (E2B). (b) Graphical representation of espin2B Actin binding domains as well as the mutations that disrupt each domain, with the E2B‐L145A mutation disrupting the xAb site, the E2B‐3LA disrupting the WH2 site and E2B‐3 W disrupting the ABM site (See text and Figure S1). (c‐f) Spectrofluorimetric pyrene‐Actin polymerization assessing the ability of E2B‐3LA (c), E2B‐L145A (d), E2B‐3 W and E2B‐3 W + 3LA (e), and E2B‐3 W + L145A (f) to provide anti‐capping effect against CP
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