Whirlin interacts with espin and modulates its actin-regulatory function: an insight into the mechanism of Usher syndrome type II

Abstract

Whirlin mutations cause retinal degeneration and hearing loss in Usher syndrome type II (USH2) and non-syndromic deafness, DFNB31. Its protein recruits other USH2 causative proteins to form a complex at the periciliary membrane complex in photoreceptors and the ankle link of the stereocilia in hair cells. However, the biological function of this USH2 protein complex is largely unknown. Using a yeast two-hybrid screen, we identified espin, an actin-binding/bundling protein involved in human deafness when defective, as a whirlin-interacting protein. The interaction between these two proteins was confirmed by their coimmunoprecipitation and colocalization in cultured cells. This interaction involves multiple domains of both proteins and only occurs when espin does not bind to actin. Espin was partially colocalized with whirlin in the retina and the inner ear. In whirlin knockout mice, espin expression changed significantly in these two tissues. Further studies found that whirlin increased the mobility of espin and actin at the actin bundles cross-linked by espin and, eventually, affected the dimension of these actin bundles. In whirlin knockout mice, the stereocilia were thickened in inner hair cells. We conclude that the interaction between whirlin and espin and the balance between their expressions are required to maintain the actin bundle network in photoreceptors and hair cells. Disruption of this actin bundle network contributes to the pathogenic mechanism of hearing loss and retinal degeneration caused by whirlin and espin mutations. Espin is a component of the USH2 protein complex and could be a candidate gene for Usher syndrome.

Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities

“…USH genes are expressed in various tissues [69–74]. This section addresses their protein expressions in the inner ear and retina (Table 3).…”

“…Espin 1–3 isoforms, derived from alternative usage of transcriptional start sites and alternative splicing, were discovered to associate with the USH2 protein complex [74]. Espins bind to actin monomers and bundle actin filaments [130].…”

“…They colocalize partially with whirlin at the photoreceptor periciliary membrane and hair cell ankle link complexes. Interaction between espins and whirlin in vivo appears important for the maintenance of actin bundle geometry [74]. Other recent findings describe interactions of myomegalin with the SANS CEN domain [131], SPAG5 (sperm-associated antigen 5, also called astrin) with the usherin cytoplasmic region [132], and Magi2 (membrane-associated guanylate kinase inverted-2) with the SANS SAM region [133].…”

Whirlin and PDZ Domain-containing 7 (PDZD7) Proteins Are Both Required to Form the Quaternary Protein Complex Associated with Usher Syndrome Type 2

“…To generate GFP-, mCherry-, FLAG-, GST-, His-, and HA-tagged proteins, protein cDNAs were cloned in frame with tags into pEGFP-C (Clontech), pmCherry-C1 (modified from pEGFP-C1), p3XFLAG-Myc-CMV-26 (Sigma-Aldrich), pGEX4t-1 (GE Healthcare), pET28a (Novagen, Billerica, MA), and CMV-HA (Clontech) vectors, respectively. GFP-, mCherry-, GAL4 AD-, and GAL4 BD-tagged WHRN FL constructs and GAL4 AD-and GAL4 BD-tagged WHRN PDZ1ϩ2 and WHRN-C constructs were reported previously (22,27). FLAG-tagged WHRN FL cDNA was subcloned using the GFPor mCherry-tagged constructs.…”

The how and why of identifying the hair cell mechano-electrical transduction channel

“…Tip links connect the shorter rows of stereocilia to their next taller neighbor and relay mechanical forces resulting from a sheering motion between those stereocilia to the hcMET-channels. Other proteins identified include: harmonin [5], myosin VIIa [41], myosin 3 [87], whirlin [108], and numerous actin binding proteins (see review [80] and proteomics screen from chicken utricle [99]). The loss of these molecules results in compromised mechanotransduction and a deterioration or even complete loss of hearing.…”