*3.2. TRIOBP-4 as an Actin Bundling Protein in the Inner Ear*

TRIOBP-4 binds directly to F-actin, principally through its R1 repeat domain, and is found along the length of filaments [49,50]. R2 shows a considerably weaker, probably hydrophobic interaction to actin [49]. In vitro assays showed TRIOBP-4 molecules to bind actin subunits at a ratio of 1:3–1:4, and that addition of TRIOBP-4 caused actin filaments to become organized into densely packed bundles, which resembled the hair cell rootlets of the inner ear [50].

TRIOBP-4 has a very specialized expression pattern and is highly expressed in the hair cells of the inner ear [50]. These cells perform mechano-electrical transduction from the fluid motion that is induced by sound into neuronal signaling. This occurs through stereocilia, organelles containing an F-actin core, which are anchored into the cuticular plate of hair cells by rootlets, and which pivot in response to fluid motion. TRIOBP-4 is found in the upper sections of these rootlets as well as along the length of the stereocilia themselves in their actin cores [18,50]. TRIOBP-4 is also found in Deiters' cells, which support the hair cells [18]. Normally, stereocilia rootlets would form in the first 16 postnatal days of mice, however, they were not seen to form at all in mice lacking the ability to produce either TRIOBP-4 or the longer isoforms (homozygous deletion of mouse exon 6, equivalent to human exon 7, Figure 1a) [50]. While stereocilia still form, they are considerably less rigid than those of wild type animals, often being found pointing in the wrong direction, and progressively degenerate [50]. These stereocilia still react to mechano-electrical transduction, but no longer have the rigidity required to remain upright and pivot in response to sound [50]. Seemingly as a result of this, these mice are profoundly deaf [50]. It therefore appears that the actin bundling function of TRIOBP-4 is crucial for the formation of the stereocilia rootlet and forming them into tight actin bundles, which are required for their stability and rigidity [18,50].

While the known role of TRIOBP-4 as an acting bundling protein has been largely restricted, so far, to studies in the inner ear, a more general role for it is suggested by two lines of evidence. First, while *TRIOBP-4* does show a very specialized expression pattern, it is not unique to the inner ear, with its transcripts notably being highly expressed in the retina [4]. Second, knockdown of TRIOBP-4 in a pancreatic cancer cell line led to reduced filopodia formation, with TRIOBP-4 seen at actin bundles of these structures [45].
