?? Spectrin Is Required For The Maintenance Of Stereocilia Of Cochlear Hair Cells And Audiological Functions

Objective:To explore the proteins that interact with myosin VI by co-immunoprecipitation and mass spectrometry,and to explore the mechanisms of?? spectrin in the maintenance of stereocilia of cochlear hair cells.Methods:Firstly,the cochlear proteins of SD rats were extracted,and the proteins interacting with myosin VI were screened by co-immunoprecipitation and mass spectrometry.Then the function of?? spectrin was selected to explore:firstly,the spatiotemporal expressions of?? spectrin in normal C57BL/6J mice was studied using immunofluorescence and Western blot,and then?? spectrin knockout mice were constructed by CRISPR/Cas9 technique.Male and female homozygotes(Gfi1-cre^-/+;Sptan1^f/f)and their siblings(Gfi1-cre^-/+;Sptan1^f/+)were used as experimental group and control group,respectively.At postnatal day?P?30,mice were performed auditory electrophysiology,immunofluorescence,scanning electron microscopy and transmission electron microscopy to explore the changes of stereocilia and hair cells,and succinate dehydrogenase staining was used to count hair cells to explore the loss of hair cells.The apoptosis of haircells was explored by Caspase-3 staining,the expression of?? spectrin was observed to explore the interaction between?? and?? spectrin,and the function of hair cells was explored by FM1-43.Results:More than 20 proteins interacting with myosin VI were found by co-immunoprecipitation and mass spectrometry.The co-expression of?? spectrin and myosin VI was found in cuticular plate.After that,it was found that the expression of?? spectrin in normal C57BL/6J mice at P1 and P3 was significantly higher than that at P7 and P30.Knockout mice showed early hearing loss at P30,and the abnormality of stereocilia of outer hair cells increased gradually with the birth time.At P15,some outer hair cells?OHCs?showed that the rootlets of stereocilia were not developed and about 40%OHCs were absent,and about 80%of them were absent at P30.Some OHCs showed apoptosis,and the function of remaining hair cells still existed.In addition,hair cells lacking?? spectrin also lacked?? spectrin in knockout mice.Conclusion:More than 20 proteins interacting with myosin?have been found.Among them,?? spectrin is very important for the maintenance of stereocilia morphology and survival of cochlear hair cells,and the lack of?? spectrin can lead to hearing loss and abnormal stereocilia morphology.