The Mechanism Of FCHSD2 In Regulating The Formation And Maintenance Of Cell Protrusions

Actin-based.finger-like cell protrusions such as microvilli and filopodia play important roles in epithelial cells.Several proteins have been identified to regulate cell protrusion formation,which helps us to learn about the underlying mechanism of this process.FCH domain and double SH3 domains containing protein 2(FCHSD2)belongs to the FCH and Bin-Amphiphysin-Rvs(F-BAR)protein family,containing an N-terminal F-BAR domain,two SH3 domains,and a C-terminal PDZ domain-binding interface(PBI).Previously,we found that FCHSD2 interacts with WASP/N-WASP and stimulates ARP2/3-mediated actin polymerization in vitro.FCHSD2 localizes along the stereocilia,which are F-actin-based and highly specialized cell protrusions on the apical surface of inner ear hair cells.All of these suggest that FCHSD2 may play a role in regulating the formation of cell protrusions and the development or maintenance of stereocilia.In the present work,we show that F-BAR domain of FCHSD2 alone promotes two types of cell protrusion formation in cultured COS-7 cells:apical cell protrusion and lateral cell protrusion.Whereas full-length FCHSD2 can not induce cell protrusions due to self-inhibition caused by the F-BAR-SH3 interaction inside it.The combination of N-WASP and FCHSD2 can relieve some of the autoinhibition and induce the formation of apical cell protrusions.Interestingly,the F-BAR domain of FCHSD2 induces lateral cell protrusion formation independently of N-WASP.Our data suggest that FCHSD2 cooperates with CDC42 and N-WASP in regulating apical cell protrusion formation.In line with this.biochemical studies reveal that FCHSD2 and CDC42 simultaneously bind to N-WASP,forming a protein complex.Furthermore,we show that the ability of FCHSD2 to induce cell protrusion formation requires its plasma membrane-binding ability caused by F-BAR domain.Stereocilia,as a kind of highly specialized inner ear hair cell surface protrusion assembled by F-actin,its development and maintenance are strictly regulated.FCHSD2 regulates actin polymerization,cell protrusion formation and localizes along the stereocilia in mice.These results let us pay attention to whether FCHSD2 plays a role in the development or maintenance of stereocilia in cochlear hair cells.We detected and analyzed the hearing phenotype of Fchsd2 knockout mice,and found that stereocilia development is unaffected by FCHSD2 inactivation.However,stereocilia maintenance is severely affected in cochlear outer hair cells(OHCs)of Fchsd2 knockout mice,which leads to severe outer hair cell loss and late-onset,progressive hearing loss eventually.Similar to the phenotype of age-related hearing loss,Fchsd2 knockout mice show increased acoustic vulnerability.Noises cause robust stereocilia degeneration in OHCs as well as enhanced hearing threshold elevation in Fchsd2 knockout mice.Fchsd2 knockout show permanent hearing loss(PTS).By observing the hearing phenotype of the constructed Atoh1-Cre+/-;Cdc42Loxp/Loxp;Fchsd2-/-double knockout mice,it was found that loss of one copy of Cdc42 gene in a Fchsd2-/-background or loss of one copy of Fchsd2 gene in an Atoh1-Cre+/-;Cdc42loxp/loxp background results in more serious hearing loss and degeneration of stereocilia.Furthermore,Atoh1-Cre+/-;Cdc42loxp/loxp;Fchsd2-/-double knockout mice show the highest threshold elevation and the most severe degeneration of stereocilia.And the localization of FCHSD2 in Atoh1-Cre+/-;Cdc42Loxp/Loxp mice(inner ear hair cell-specific Cdc42 knockout mice)remains unchanged.stereociliaWe need to conduct more in-depth studies on Fchsd2-/-knockout mice to clarify the specific molecular mechanism of FCHSD2 in the maintenance of stereocilia.Innovation and Significance:In this project,the truncated or full-length expression vectors of FCHSD2,CDC42,and N-WASP were transfected into COS-7 cells to explore the method and possible molecular mechanism of FCHSD2 in regulating cell protrusions formation at the cellular level.We studied the effect of FCHSD2 knockout on the development or maintenance of the stereocilia at the animal physiological level through the Fchsd2 knockout mice.By constructing Cdc42/Fchsd2 double knockout mice,we analyzed whether CDC42 involved in the process of stereocilia maintaince by FCHSD2.We have the innovative research results and significance of following five points:1.In the present work,we revealed that FCHSD2 could cause two types of cell protrusion by different ways in cultured COS-7 cells.One type is apical cell protrusion,the other one is lateral cell protrusion.2.We verified that there is an interaction between the F-BAR domain and the SH3 domain in FCHSD2 for first time,which leads to self-inhibition in the full-length FCHSD2.3.We have verified for the first time that FCHSD2 and CDC42 simultaneously bind to N-WASP,forming a protein complex,and cooperatively promote the formation of filopodia-like apical cell protrusion,providing a new possibility way for the formation of filopodia.4.At the level of animal physiology,we used Fchsd2 knockout mice to study the effect of FCHSD2 deletion on the development or maintenance of hair cell stereocilia.The data showed that FCHSD2 knockout has no significant effect on the development of hair cell stereocilia,but the maintenance of outer hair cell stereocilia significant was defected severely.Fchsd2 knockout mice exhibite progressive hearing loss.And we found that Fchsd2 knockout mice are more vulnerable to noise stimulation by the experiments of noise exposure.So far.there are few studies on the function of FCHSD2 in animals.This article provides a new direction for the study of FCHSD2 function in vivo.So far.there are few studies on the function of FCHSD2 in animals.This article provides a new finding for the study of FCHSD2 in vivo function.5.We tested the more elaborated phenotype of Cdc42/Fchsd2 double knockout mice.and found that FCHSD2 and CDC42 cooperate to regulate auditory function in mice.