Rapid Turnover of Stereocilia Membrane Proteins: Evidence from the Trafficking and Mobility of Plasma Membrane Ca2+-ATPase 2

Abstract

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca 2ϩ ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of ϳ5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 m 2 /s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that ϳ60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 m 2 /s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.

FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing

“…2C) and in the stereociliar plasma membrane. This profile is very similar to that of calcium-ATPase2, previously shown to localize at the plasma membrane of stereocilia (5). Mutations in the gene encoding for this transporter cause deafness in mice (6), and have been reported to be a genetic modifier of hearing loss in humans (7).…”

“…Evidence supporting a critical role for FAM65B in hearing includes a mutation coinherited with hearing loss in a large kindred, expression of Fam65b in the cochlea of embryonic and adult mouse, localization of fam65b to the zebrafish otic vesicle, and impairment of hearing by gene knockdown in zebrafish. Furthermore, FAM65B localizes to the plasma membrane of the stereocilia hair bundle, the essential cellular organelle in which electrical signals originate and from which they are transmitted after a mechanical stimulus (5). Fam65b transcripts are detected early during the development of the cochlea, a stage in which actin-filled microvilli at the apex of hair cells start to rearrange and elongate.…”

A New Compartment at Stereocilia Tips Defined by Spatial and Temporal Patterns of Myosin IIIa Expression

“…Although some stereocilia membrane proteins can diffuse laterally to distribute along the stereocilia membrane (Grati et al, 2006), other proteins including components of the tip link complex may require active transport. Myosin XVa has been shown to shuttle the PDZ protein whirlin in stereocilia and to undergo intrafilopodial motility in transfected cultured cells (Belyantseva et al, 2005).…”

The role of central nervous system plasticity in tinnitus

“…Eggermont and Roberts (2004) recently summarized the literature and with exception of the case where high doses of salicylate were administered (Müller, Klinke, Arnold, & Oestreicher, 2003), the universal outcome in auditory nerve fibers from overstimulation or ototoxic treatment is reduced sound-driven activity and a reduction (or no change) in spontaneous activity (Kiang, Moxon, & Levine, 1970;Liberman and Kiang, 1978;Mulheran, 1999;Stypulkowski, 1990). Even the suggestion that stereocilia cytoskeletal damage is a contributing factor becomes less compelling in the light of recent evidence that the crystalline matrix of stereocilia undergo a continuous renewal (Grati et al, 2006;Rzadzinska et al, 2004;Schneider, Belyantseva, Azevedo, & Kachar, 2002), and that losses in stereocilia stiffness can rapidly recover (Saunders and Flock, 1986;Szymko et al et al, 1995;Duncan and Saunders, 2000). Perhaps the most important observation that tinnitus has a retrocochlear origin is the observation that tinnitus persists after surgical resection of the auditory nerve (House and Brackman, 1981).…”