Hearing depends upon sound-induced deflections of mechanosensory stereocilia, actin-based microvilli-like projections on the apical surface of each cochlear hair cell organized into ranks of increasing height (). Nanometer-scale deflections tension the tip links between stereocilia and gate cation-selective mechanotransduction channels present on all but the tallest stereocilia (Beurg et al., 2009
). The mechanical properties of each stereocilium must be precisely tuned for optimal sensitivity.
Stereocilia Rootlets within the Organ of Corti and TRIOBP Structure, Isoforms and Immunogens
Mammalian stereocilia contain a core of uniformly-spaced polarized actin filaments inter-connected with espin and fimbrin/plastin (reviewed in Frolenkov et al., 2004
). The barbed ends of the filaments are oriented toward the stereocilia tips, a site of actin monomer addition (Schneider et al., 2002
). These filaments form a paracrystalline array that confers rigidity and allows each stereocilium to act as a stiff lever. When deflected, stereocilia pivot about their insertion points near the apical surface of the cell where the diameter of stereocilia tapers (Crawford et al., 1989
; Karavitaki and Corey, 2006
). Actin filament topology within the taper differs from the main stereocilia core. In this region, transmission electron microscopy (TEM) reveals a rootlet; an electron dense structure that penetrates into the cell body and also extends a comparable distance into the stereocilia core (Flock and Cheung, 1977
) (). Similar rootlet structures were observed at the base of intestinal microvilli (Matsudaira and Burgess, 1982
). Rootlets were proposed to anchor stereocilia into the actin-rich meshwork of the cuticular plate and/or provide flexible elements for durable pivoting of stereocilia about their tapers (Furness et al., 2008
; Tilney et al., 1983
; Tilney et al., 1986
). However, in the absence of experimental models, the role of rootlets in hair bundle micromechanics and the molecules that guide their development remain elusive.
Here we show that TRIOBP is an actin-bundling protein that is critical for rootlet formation. Mutations of human TRIOBP
, a gene encoding multiple isoforms, are associated with profound, prelingual deafness DFNB28 (MIM #609823) (Riazuddin et al., 2006
; Shahin et al., 2006
). The alternative splice isoforms of TRIOBP are produced through the use of two alternate promoters and can be grouped into three classes (). The first are long transcripts that utilize a distal promoter upstream of exon 1 and terminate in exon 24, which encodes TRIOBP-5 (~218-kDa) in humans (). The second class is initiated from the same promoter but terminates immediately after exon 6, and encodes a shorter protein product, TRIOBP4 (~107-kDa) that contains the repeat motifs of exon 6 but none of the carboxy domains of TRIOBP-5. The third class, represented by TRIOBP-1 (~72-kDa; Seipel et al., 2001
) is initiated from a promoter downstream of exon 6. TRIOBP-1 encodes a protein that does not contain the N-terminal internal repeat motifs, but does include the carboxy domains of TRIOBP-5 encoded by exons 11–24 (). Thus, TRIOBP-1 and TRIOBP-4 share no exons or amino acid coding sequence. Over-expression studies have suggested that TRIOBP-1 (previously named TARA) binds and stabilizes the actin cytoskeleton in HeLa cells (Seipel et al., 2001
). While TRIOBP-1 is ubiquitous, TRIOBP-4 and TRIOBP-5 are expressed predominantly in the eye and inner ear (Riazuddin et al., 2006
; Shahin et al., 2006
). To date, all of the mutations of TRIOBP
causing human deafness DFNB28 are located in exon 6 (), and only affect TRIOBP-4 and TRIOBP-5 (TRIOBP-4/5).
All three isoform classes of TRIOBP localized to the stereocilia rootlets of inner ear hair cells. In vitro, purified TRIOBP-4 organizes actin filaments into bundles of unusually high density that resembled stereocilia rootlets. We engineered a TRIOBP-4/5 deficient mouse recapitulating human DFNB28 deafness. In this mouse, rootlets fail to develop resulting in stereocilia that were abnormally flexible at the pivot points and easily damaged by over-stimulation. Thus, the bundling of actin filaments by TRIOBP is essential for the biogenesis of rootlets that provide durable flexibility at the taper and mechanical rigidity to the stereocilia bundle.