Molecular Mechanism Underlying Stereocilia Development Mediated By Liquid-Liquid Phase Separation Of The Tip Complex Density

The human auditory system consists of the outer ear,middle ear,inner ear and auditory nervous system.The essence of hearing generation is the process of converting the mechanical vibration of sound waves into electrical signals.The stereocilia of the hair cells in the inner ear Corti’s organ play a key role in this process.Stereocilia are specialized hair-like structures at the apical surfaces of hair cells,which are formed by cross-linking of highly organized parallel actin filaments(F-actin).Stereocilia are organized into three rows of graded height after maturation.The mechanoelectrical ion channels are located at the tip of shorter stereocilia but not the tallest stereocilia.Neighboring stereocilia are interconnected by tip links.Mechanical force evoked by sound waves results in stereocilia deflection toward the tallest row,which in turn activates the mechanoelectrical transducer channels located in the shorter stereocilia,thus achieving the mechano-electrical transduction and auditory perception.Therefore,proper development and maintenance of stereocilia are essential for normal hair cell functions.However,it still remains unclear how stereocilia adopt such a staircase-like architecture.Transmission electron microscope images have shown that there is an electron-dense region at the tips of stereocilia,which is termed as tip complex density(TCD).TCD is enriched with a large number of proteins,including Whirlin,Eps8,Eps8L2,Myo 15a,Gpsm2,Gαi,etc.These proteins form a complex interaction network.It was reported that mice deficient in any of the genes encoding these proteins show extremely short stereocilia and profound deafness.In these abnormal stereocilia,the distribution of TCD is obviously patchy and irregular,as well as accompanied by the decrease of F-actin bundles.A large number of mutations in the above-mentioned genes have been identified in patients with hearing loss syndrome such as Usher syndrome,suggesting that proper assembly of TCD protein complexes plays a critical role in the growth and development of stereocilia.More interestingly,Gpsm2 and Gai are specifically located at the TCD of the first row ofstereocilia,which is considered to be a key determinant for the row identity of the tallest stereocilia.Therefore,the assembly of the Gpsm2-Gαi-mediated TCD complex at the tallest stereocilia may be the core mechanism of the staircase-like arrangement of stereocilia.Notably,this dense structure exhibits properties reminiscent of the postsynaptic density(PSD)in neuron which is demonstrated to form via liquid-liquid phase separation(LLPS)of key components among the PSD.Therefore,we propose the scientific hypothesis that the assembly of stereocilia TCD may be driven by LLPS mediated by multivalent interactions among the TCD protein complexes,which may underly the molecular mechanisms that regulate the growth and staircase-like arrangement of stereocilia.In this thesis,through combination of diverse approaches including molecular biology,biochemistry,structural biology and cell biology,we systematically studied the interactions among TCD complexes and determined crystal structures of WhirlinMyo15a and Gpsm2-Whirlin complexes which revealed the detailed molecular mechanisms of the assembly of the TCD protein complexes.Importantly,we found that Whirlin and Gpsm2 undergo LLPS both in living cells and in vitro.The specific multivalent interactions among Whirlin-Myo15a-Eps8 promote the LLPS formation and lead to the formation of TCD-like condensates.The TCD-like condensates reconstituted in vitro can effectively promote the F-actin bundling.A deafnessassociated mutation of Myol5,p.S3525AfsX29,interferes with the condensates formation and consequently impairs actin bundling.Furthermore,we found that Gpsm2-Gai can further promote the ability of whirlin-Myo15a-Eps8 to form LLPS,and the five-component TCD condensates mediated by LLPS displays much stronger Factin bundling ability than the whirlin-Myol5a-Eps8 condensates did.The study confirmed that this promotion effect depends on the LLPS of Gpsm2.A deafnessassociated mutation of Gpsm2,p.R318RfsX8,leads to impaired formation of the 5xTCD condensates and consequently reduced actin bundling.Taken together,we elucidated the interaction mechanism and structural basis of multiple TCD protein complexes at the tips of stereocilia.We revealed for the first time the molecular mechanism underlying the assembly of stereocilia TCD mediated by LLPS which ensures local enrichment of high concentration of actin regulatory factors(such as Eps8)to promote F-actin bundle,thus regulating the development of stereocilia.We further demonstrated that Gpsm2-Gai,which is specifically located at the tallest stereocilia,promotes the assembly of TCD like condensates through LLPS formed by Gpsm2,thereby promoting F-actin bundle more efficiently at the tallest stereocilia and defining the row identity of the tallest stereocilia.This work provides valuable molecular mechanisms for the development and staircase pattern of stereocilia,and offers possible explanations for the pathogenesis of hearing loss related diseases.