Roles of the Espin Actin-Bundling Proteins in the Morphogenesis and Stabilization of Hair Cell Stereocilia Revealed in CBA/CaJ Congenic Jerker Mice

Abstract

Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10–0.14 µm, making stereocilia ∼30%–60% thinner than wild type and suggesting that they contained ∼50%–85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

“…4 A and Table 1). The increased difference in the P9 OHC bundles reflected the fact that the height of the tallest OHC stereocilia did not change in Myo3a −/− Myo3b −/− mice between P0 and P9, whereas it decreased by ∼36% in the control mice, as expected (Sekerková et al, 2011;Fig. 4 A).…”

“…3 B), and the existence of transient thin outgrowths at the very tips of stereocilia in a few Myo3a +/− Myo3b −/− IHCs on P0 (Fig. S3 C), similar to the transient "candlewick-like" tapering reported in the vestibular stereocilia of je/+ mice (Sekerková et al, 2011). Thus, subtle functional interactions between class III myosins and espin-1 might take place in the early developing cochlear hair bundles.…”

“…A second phase of selective stereocilia development extends up to the first two postnatal weeks, during which the three tallest stereocilia rows continue to widen and change length until they reach their mature size (in the mouse cochlea, they shorten except in the most apical cochlear regions, where they elongate; Sekerková et al, 2011), whereas the remaining microvilli regress (Barr-Gillespie, 2015; Fig. 3 B, top).…”

“…Also consistent with the proper targeting of espin-1 in the absence of myosins IIIa and IIIb, a cochlear phenotype opposite to that of Myo3a −/− Myo3b −/− mice is observed in the Jerker (je/je) mice that carry a mutation inactivating the gene coding for all espin isoforms (Sekerková et al, 2004). Jerker mice have abnormally thin and short stereocilia from P0 onwards in both IHCs and OHCs, but the formation of the initial U or V bundle shapes and the first phase of stereocilia elongation appear unaffected (Sekerková et al, 2011). This suggests that the early role of class III myosins in hair bundle morphogenesis is in fact independent of espins.…”

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

“…Stereociliary growth and maintenance involves actin-binding proteins such as espin (7,8), plastin (9), twinfilin 2 (10), gelsolin (11), and unconventional myosin motors including myosin XVa (12) and myosin IIIa (13). Currently, we do not have a complete molecular understanding of hair bundle structure or how its growth and maintenance are controlled.…”