Structural And Functional Studies Of Tip-link Complex In Stereocilia/microvilli

Stereocilia locate at the apical surface of hair cells in cochlea,responsible for transforming the mechanical signal(sound wave)to electrical signal(membrane potential caused by opening of the ion channels and depolarization of the cells).Microvilli locate at the outer surface of epithelial cells of small intestine,responsible for increasing cell surface for nutrient uptake.Despite their differences in morphologies(stereocilia form a staircase-like structure whereas microvilli forming a densely packed structure),they are both actin filaments-based subcellular structures.Developmental defects or morphological changes of these structures are closely related to inherited diseases such as deafness,blindness and enteropathy.Recent studies demonstrated that despite clear morphological and functional differences,the protein complexes controlling their development and organization are highly similar.The complex in stereocilia is called Tip-link complex,while that in microvilli is referred to as intermicrovillar adhesion complex(IMAC).Harmonin is a protein containing multiple PDZ domains.It is a central component of both the complexes and required for the development and maintenance of hair cell stereocilia and brush border microvilli.Mutations in the Harmonin-encoding gene USH1 C can cause Usher syndrome type 1C(USH1C)disease.The cadherin-related family member 2(CDHR2)is involved in the assembly of adjacent microvilli through the formation of adhesion complexes.A recent study showed that CDHR2,uses the PDZ-binding motif(PBM)in its cytoplasmic tail to bind to the PDZ2 and coiled-coil(CC)domain(PDZ2CC)of Harmonin.However,the structural basis of the interaction and the mechanism for Harmonin mutations causing USH1 C disease are unclear.In addition,the L48 P mutation found in the N-terminal Ankyrin repeats of Sans in stereocilia tip-link can cause Usher syndrome type 1G(USH1G)disease.And L48 P mutation of ANKS4 B protein,its close homolog in microvilli,also resulted in defective microvilli location.Again,it is unclear about the N-terminal Ankyrin repeats structures of Sans and ANKS4 B and the mechanism of Sans protein mutation causing USH1 G disease.Sans can also function in the nuclear,while which domain of Sans participates in this is unknown.To better understand the assembly and functional mechanism of the tip-link complex,our research is divided into two parts,and the results are as follows:(1)Structure of the Harmonin PDZ2 and coiled-coil domains in a complex with CDHR2 tail and its implications in Usher syndrome.We solved the structure of Harmonin PDZ2 CC domain and CDHR2 PBM complex by X-ray crystallography.The structure showed that PDZ2 together with its C-terminal short α-helix extension bound to CDHR2 PBM in a typical way.Mutations of Harmonin found in patients could affect its PBM binding capability,as confirmed by our biochemical approaches.Furthermore,the structure,together with the in vitro mutagenesis experiments,indicated that CC might form an antiparallel twisted dimer under high concentration,a concentration that can be reached when Harmonin underwent liquid–liquid phase separation,a mechanism in which biological macromolecules participate in cell function by forming high concentration droplet-like concentrate.Our study provided the first glimpse at the biochemical and structural properties of the CC domain and will be valuable for further investigation of the physiological and pathological roles of Harmonin.(2)Structure and function of Ankyrin repeats for Sans and ANKS4 B.Firstly,we got the high-resolution structures of the Ankyrin repeats of Sans and ANKS4 B by X-ray crystallography.Mutations(L48P,L84P)on Sans in USH1 G patients can decrease the protein stability as confirmed by our biochemical approaches.Furthermore,our cell biology studies found that Sans protein could be located not only in the cytoplasm but also in the nucleus.In contrast,ANKS4 B was exclusively distributed in the cytoplasm.We further generated Sans/ANKS4 B chimeric proteins by exchanging N-terminal Ankyrin repeats to confirm that the importance of N-terminal Ankyrin repeats to the nuclear location of Sans.Moreover,mutagenesis experiments showed that the three disease-related sites(R10,S119,R146)on Sans Ankyrin repeats may play a key role in nuclear location.Our work would be valuable to guide future studies on the molecular mechanism of USH1 G disease and the potential binding partners of their Ankyrin repeats of Sans and ANKS4 B.In summary,we hope that our discoveries will provide insights into the molecular basis of how stereocilia/microvilli develop and maintain,elucidate the disease-causing mechanism of inherited haring loss or enteropathy,bridge the gap between genetic mutations and clinical symptoms,and finally benefit the future treatments of such diseases.