James R. Bartles

The Deaf Jerker Mouse Has a Mutation in the Gene Encoding the Espin Actin-Bundling Proteins of Hair Cell Stereocilia and Lacks Espins

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.

Parallel actin bundles and their multiple actin-bundling proteins.

Parallel actin bundles are present in a diverse array of structures, where they are critical determinants of cellular shape and physiology. In the past 18 months, new findings have solidified the concept that parallel actin bundles are assembled in cells through the sequential action of multiple actin-bundling proteins and have begun to shed light on the roles played by the individual actin-bundling proteins.

Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo.

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.

Actin-Bundling Protein TRIOBP Forms Resilient Rootlets of Hair Cell Stereocilia Essential for Hearing

Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets but distinct from bundles formed by espin, an actin crosslinker in stereocilia. We generated mutant Triobp mice (Triobp(Deltaex8/Deltaex8)) that are profoundly deaf. Stereocilia of Triobp(Deltaex8/Deltaex8) mice develop normally but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.

Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key?

Abstract The Madin-Darby canine kidney (MDCK) cell sorts newly synthesized apical and basolateral plasma membrane proteins intracellularly and then ships them directly to the correct plasma membrane domains, whereas the rat hepatocyte sorts apical and basolateral proteins after their arrival at the basolateral domain of the plasma membrane and then employs a transcytotic mechanism to deliver the apical proteins to the apical domain. This difference in plasma membrane protein sorting pathways may relate to the observation that the hepatocyte, unlike the MDCK cell, does not have an apically directed secretory pathway.

The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Varitint-waddler (Va and VaJ) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, VaJ, has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd3+, have a conductance of 50–70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and VaJ mice.

Sertoli Cell Ectoplasmic Specializations in the Seminiferous Epithelium of the Testosterone-Suppressed Adult Rat

Abstract The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.

Espins Are Multifunctional Actin Cytoskeletal Regulatory Proteins in the Microvilli of Chemosensory and Mechanosensory Cells

Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells, and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca2+-resistant manner, bound actin monomer via a WASP (Wiskott-Aldrich syndrome protein) homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53, and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5-bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics, and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in various mechanosensory and chemosensory cells.

125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination.

Two substantial improvements in sensitivity in the identification of 125I-wheat germ agglutinin-binding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% (w/v) polyvinylpyrrolidone (average Mr 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with 125I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of 125I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mM sodium metaperiodate followed by 5 mM aniline in the presence of 30 mM sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.

Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice.

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.