Whirlin Interacts With Espin And Modulates Its Actin-regulatory Function

Usher syndrome is a disease affecting both vision and hearing. It isclassified into three clinical types, with Type II being the most common formaccounting for about70%of all cases. Genetic defects in three genes (USH2A,USH2C and USH2D) are known to underlie this type of Usher syndrome. Thiscomplex is localized mainly at the periciliary membrane complex inphotoreceptors and the ankle-link of the stereocilia in hair cells. Many proteinshave been found to interact with USH2proteins in vitro, suggesting that theyare potential additional components of this USH2complex and that the genesencoding these proteins may be the candidate USH2genes. However, thebiological function of this USH2protein complex is largely unknown.In this study, we identified the direct interaction between espin and theprotein encoded by the USH2D gene, whirlin. We showed that this interactionis important for the architecture of actin bundles cross-linked by espin. Ourdemonstration of espin expression in photoreceptors and espin expressionchanges in the whirlin knockout mouse, an animal model for Usher syndrome,is significantly novel, which provide the first clue to the molecular defects,other than the defects in the USH2proteins, in the Usher type II pathogenesis.Based on the predicted functional domains in USH2proteins, their cellularlocalizations in photoreceptors and hair cells, the observed phenotypes inUSH2mutant mice, and the known knowledge about diseases similar to USH2,putative biological functions of the USH2complex have been proposed. Finally,therapeutic approaches for this group of diseases are now being activelyexplored.Method: 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 confirmedby their coimmunoprecipitation and colocalization in cultured cells. Thisinteraction involves multiple domains of both proteins and only occurs whenespin does not bind to actin. Espin was partially colocalized with whirlin in theretina and the inner ear. In whirlin knockout mice, espin expression changedsignificantly in these two tissues. Further studies found that whirlin increasedthe motility of espin and actin at the actin bundles cross-linked by espin and,eventually, affected the dimension of these actin bundles. In whirlin knockoutmice, the stereocilia was thickened in inner hair cells.Results:1.Whirlin interacts with espinA yeast two-hybrid screen is used to further confirm the interactionbetween whirlin and espin, we did coimmunoprecipitation using recombinantwhirlin and GFP-fused espin in HEK293cells. The whirlin antibody canprecipitate GFP-fused espin in cells transfected with GFP-fused espin andrecombinant whirlin. A careful examination of the two protein signals revealedtheir partial colocalization. The correlation of the signal intensities from thetwo proteins in the same pixel was very strong in the region of cytoplasm withno thick espin fibers. The Pearson's coefficients in these regions are in therange between0.7and0.9. In summary, the partial colocalization betweenwhirlin and espin, shown here, suggests that whirlin and espin interact witheach other and that not all of these two proteins participate in their interaction.In whirlin-transfected cells, whirlin did not colocalize with actin filamentsstained with phalloidin. In addition, whirlin was not coimmunoprecipitatedwith actin from whirlin-transfected cells (data not shown) or the mouse retina.These results demonstrated that whirlin had no actin-binding activity. In espin-transfected cells, espin fibrous structures were in fact actinbundles as revealed by phalloidin staining, indicating that they are espincross-linked actin bundles. In espin and whirlin double transfected cells, theespin fibrous structures remained stained with phalloidin. However, whirlinshowed very weak signals along these espin/actin bundles. This findingsuggested that whirlin may not interact with espin when espin cross-linkedactin filaments. We found that cytochalasin D treatment before immuno-precipitation, which is thought to release espin when the actin bundles weredisrupted, increased fourfold the amount of GFP-fused espin in the whirlinimmunoprecipitate. This result is consistent with our colocalization findingand supports the notion that theinteraction between whirlin and espin occurs when espin does not bundle theactin filaments.2.Multiple regions of whirlin and espin are involved in their interactionSupportively, individual coimmunoprecipitations of espin AR and PR withwhirlin were found in HEK293cells. These two espin fragments also showedcolocalization with whirlin in the transfected cells Surprisingly, to somedegree, espin ABM also displayed colocalization with whirlin in their doubletransfected cells. This suggested that espin ABM was likely to directly orindirectly associate with whirlin as well. Because both espin WH2H and WH2Lfragments did not colocalize with whirlin, the espin WH2domain couldprobably inhibit the association between espin ABM and whirlin.3.Partial colocalization of whirlin and espin is observed in photoreceptorsand hair cellsImmunostaining of the lightly fixed mouse retina detected espin proteinsignals in the inner segment and the synaptic end of photoreceptors (the innersegment layer and the outer plexiform layer, respectively). At the highmagnification, weak bar-shaped espin signals were seen at the top of the inner segment. In hair cells, double staining localized espin along the entirestereocilia and whirlin at the tip and the ankle-link (the base) of the stereocilia.The two proteins were colocalized at the ankle-link but not the tip of thestereocilia. Taken together, the partial colocalization between whirlin and espinindicates that the interaction between the two proteins exists in photoreceptorsand hair cells.4.Espin expression is altered in the whirlin knockout retina and inner earCompare to wild type mice retinas, in whirlin knockout retinas, theexpression level of espin3decreased to about10%, while that of espin4increased by about ten fold. Theexpression of espin1did not change significantly. As we already showedabove, whirlin was able to interact with espins1-3. In the whirlin knockoutouter hair cells, the intensity of espin signals significantly decreased to about30%of that in the wild-type. Therefore, the alteration of espin expression in theretina and the hair cells in the absence of whirlin strongly suggests that the twoproteins interact in vivo in these tissues.5.Whirlin accelerates the espin motility at the actin bundle in filopodia andwhirlin accelerates the actin motility at the actin bundle cross-linked byespinWe conducted fluorescence recovery after photobleaching (FRAP) usingthe GFP-tagged espin. Whirlin was found to significantly shorten the espinrecovery time but had no effect on espin final recovery percentage at thefilopodia. This suggests that whirlin accelerates the exchange between pools offree espin and espin bound with actin bundles at the filopodia and, thus, mayindirectly weaken the cross-link of espin with actin filaments.To further investigate the whirlin effect on actin bundles cross-linked byespin, we performed FRAP using GFP-tagged actin. When whirlin wascotransfected with both GFP-actin and espin, the recovery time was shortened by half and the final recovery percentage stayed the same at the intracellularthin fibers,compared to those in cells transfected only with GFP-actin andespin.6. Ablation of whirlin causes thick stereocilia in the inner hair cellsTo elucidate the potential consequence of changes in espin expression inwhirlin knockout mice, we measured the diameter of the hair cell stereociliaand discovered that the whirlinknockout mice had thick stereocilia in the inner hair cell and normal stereociliain the outer hair cell. We propose that both espin and whirlin are required tomaintain the normal diameter of stereocilia in the wild-type mice and that theyplay opposite roles in this function.Conclusion:We conclude that the interaction between whirlin and espin and thebalance between their expressions are required to maintain the actin bundlenetwork in photoreceptors and hair cells. Disruption of this actin bundlenetwork contributes to the pathogenic mechanism of hearing loss and retinaldegeneration caused by whirlin and espin mutations. Espin is a component ofthe USH2protein complex and could be a candidate gene for Usher syndrome.