The espins are actin-bundling proteins of brush border microvilli and Sertoli cell-spermatid junctions. We have determined that espins are also present in hair cell stereocilia and have uncovered a connection between the espin gene and jerker, a recessive mutation that causes hair cell degeneration, deafness, and vestibular dysfunction. The espin gene maps to the same region of mouse chromosome 4 as jerker. The tissues of jerker mice do not accumulate espin proteins but contain normal levels of espin mRNAs. The espin gene of jerker mice has a frameshift mutation that affects the espin C-terminal actin-bundling module. These data suggest that jerker mice are, in effect, espin null and that the jerker phenotype results from a mutation in the espin gene.

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at ∼1.5 s−1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.

An ∼30-kD isoform of the actin-binding/ bundling protein espin has been discovered in the brush borders of absorptive epithelial cells in rat intestine and kidney. Small espin is identical in sequence to the COOH terminus of the larger (∼110-kD) espin isoform identified in the actin bundles of Sertoli cell–spermatid junctional plaques (Bartles, J.R., A. Wierda, and L. Zheng. 1996. J. Cell Sci. 109:1229–1239), but it contains two unique peptides at its NH2 terminus. Small espin was localized to the parallel actin bundles of brush border microvilli, resisted extraction with Triton X-100, and accumulated in the brush border during enterocyte differentiation/migration along the crypt–villus axis in adults. In transfected BHK fibroblasts, green fluorescent protein–small espin decorated F-actin–containing fibers and appeared to elicit their accumulation and/or bundling. Recombinant small espin bound to skeletal muscle and nonmuscle F-actin with high affinity (K d = 150 and 50 nM) and cross-linked the filaments into bundles. Sedimentation, gel filtration, and circular dichroism analyses suggested that recombinant small espin was a monomer with an asymmetrical shape and a high percentage of α-helix. Deletion mutagenesis suggested that small espin contained two actin-binding sites in its COOH-terminal 116–amino acid peptide and that the NH2-terminal half of its forked homology peptide was necessary for bundling activity.

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Varitint-waddler (Va andVaA very common cause of deafness is the loss of hair cells, which degenerate because of environmental factors or genetic mutations. Varitint-waddler (Va) mice have a mutation that causes an alanine-to-proline substitution (A419P) in the fifth transmembrane domain of TRPML3, a presumed ion channel. A second allele, Va J , contains the A419P mutation in cis to an I362T mutation (1). The earliest sign of inner ear damage in Va mice is hair cell degeneration, which begins in embryogenesis as hair cells differentiate and continues postnatally. During degeneration, the hair cells bulge out of the apical side of the epithelium and become extruded. Eventually these mice also develop abnormalities in supporting cells, in the tectorial membrane, and in the stria vascularis and in the endocochlear potential that they help generate. The defects of Va J mice are similar although less severe, suggesting that the mutation I362T attenuates the effects of A419P (1-3). A comprehensive examination of TRPML3 expression in inner ear has not been published. Expression of TRPML3 protein in hair cells was suggested by immunocytochemical methods, although results for other inner ear cell types were not reported (1).Mutations in two other TRP channels, Drosophila TRP and human TRPML1 (mutations in which cause mucolipidosis type IV), are associated with degeneration of the retina. In both cases, the channels seem necessary for cellular viability, because recessive, loss-of-function mutations cause degeneration (4, 5).By contrast, the Va and Va J mutations in TRPML3 are semidominant, although it remains unclear whether this is due to gain-of-function, haploinsufficiency, or dominant-negative effects (1, 6). ResultsWe first established which cells expressed TRPML3 in the inner ear of wild-type mice. In situ hybridization on mouse inner ear using probes from two nonoverlapping regions of TRPML3 mRNA gave identical results [ Fig. 1 and supporting information (SI) Fig. 6]. TRPML3 mRNA was most abundant in the marginal cells of the cochlear stria vascularis and the dark cells of the vestibule (Fig. 1 i, l, and m and SI Fig. 6 i, l, and m). Both cell types are involved in producing the endolymph and therefore the endocochlear potential (7-9). This expression pattern is in keeping with the reduced stria vascularis and the rounding up and loss of the cytoplasmic processes of its marginal cells in Va/Va and Va/ϩ mice (2) and with the reduced endocochlear potentials of the Va J mutants (1, 3).In addition, other cells that line the cochlear scala media and the connected vestibular endolymphatic spaces expressed TRPML3 mRNA, including (i) supporting cells at the marginal part of the lesser epithelial ridge (Hensen and Claudius cells), which in Va and Va J mice (homozygotes and heterozygotes) appear undifferentiated or degenerate ( Fig. 1i and SI Fig. 6i); (ii) cells of the spiral limbus that produce the tectorial membrane, which in Va mice develops abnormally and fails to attach to the reticular lamina ( Fig. 1i and SI Fig. 6i...