Purpose:  Whirlin mutations cause retinal degeneration and hearing loss in Usher syndrome type II (USH2) and nonsyndromic deafness, DFNB31.Usher syndrome is the most common genetic cause for the combined vision and hearing loss. Among its three clinical types, type II (USH2) is predominant and accounts for about 70% of all Usher cases. It is manifested as retinitis
pigmentosa and congenital moderate hearing loss. Currently, Whirlin is a causative gene of USH2. It can recruit the other two proteins.  However, the biological function of this
complex is little known. we identified espin, an actin-binding/bundling protein involved in human deafness when defective, as a whirlin-interacting protein.  In addition, the interaction between whirlin and espin occurs when espin doesn’t bind to the actin filaments.

Methods:  We transfected the Cos-7 cells with whirlin and espin with different tags.The confocal images shows these two proteins partial colocalized. In vinculin and actin double transfected cell, protocol is performed above similarly.

Transfected HEK293 cells were collected from culture medium.  Cytochalasin D treatment was performed before coimmunoprecipitation.
Results: We transfected whirlin and espin with different tags to the Cos-7 cells. These two proteins colocalized at the cytoplasm.  A careful examination of the two protein signals revealed their partial colocalization. In summary, the partial colocalization between whirlin and espin, shown here, suggests that whirlin and espin interact with each other and that not all of these two proteins participate in their interaction.

 In espin and whirlin double transfected cells,whirlin showed very weak signals along these espin/actin bundles, suggesting that the interaction between whirlin and espin occurs when espin doesn’t bind to the actin filaments. We added cytochalasin D before immunoprecipitation, and found intensity of gfp-fused espin increased fourfold in the whirlin immunoprecipitate.
The interaction between these two proteins was confirmed by their coimmunoprecipitation and colocalization in cultured cells. Whirlin exhibited an inhibitory role against the actin binding/bundling activity of espin.

 In theCos-7 cell transfected with vinculin and actin, we found vinculin is colocalize with the end of actin filament. binding to the barbed end, fast growing end ofactin filaments.

Conclusions:Interaction between whirlinand espin occurs when espin doesn’t bind to the actin filaments. Whirlin
exhibited an inhibitory role against the actin binding/bundling activity ofespin.