| BACKGROUND AND OBJECTIVEInner hair cells are the auditory receptor cells. Inner hair cells play irreplaceable roles in auditory maintenance, because they can transform the mechanical stimuli into nerve impulse. Stereocilia, microvilli-like protrusions on the hair cells’ apical surface, plays an important role in the mechanical-electrical energy conversion process. Therefore, the development and maintenance of stereocilia are great significance on auditory conduction. The stereociliary rootlets are inserted into the cuticular plate of hair cells. Cuticular plate is a dense structure formed by filamentous actin (F-actin), locates on the hair cell cytoplasmic surface and plays an important role as an anchor for stereocilia. However, the mechanism about how development and maintenance of the cuticular plate are poor understood.Stereocilia is mainly based on F-actin. The length of the stereocilia is unchanged but the F-actin core constantly updates itself:new actin monomers increase from the top of stereocilia, and old actin monomers are dissociated from the root of stereocilia. During the development of stereocilia, the polymerization speed of actin monomers is faster than the dissociation speed, so that the stereocilia are growing. After being mature, the polymerization speed of actin monomers is the same as the dissociation speed, so that the length of stereocilia remains unchanged.Except F-actin, there are many other stereociliary proteins, such as Myo15a, Whirilin, Twinfilin 2 and so on, localizing in different locations in the stereocilia. These proteins play important roles in the development of stereocilia and in the auditory process. Development and maintenance of stereocilia is a complex and precise process. If an exception occurs in the process, the stereociliary structure will be damaged and it will lead to syndromic or nonsydromic deafness, which could seriously affect the quality of patients’ life. At present, the mechanism of stereociliary development and maintenance is remains unclear. Other stereociliary proteins involved in this process will be found. These problems prevent us from providing the theoretical basis for clinical research.We found that FCHSD1 and FCHSD2 expressed in inner hair cells. FCHSD1 was detected in the stereocilia and in the cuticular plate, whereas FCHSD2 mainly localized along the stereocilia in a punctuate pattern. FCHSD1 and FCHSD2 are the mammalian orthologs of Drosophila Nervous Wreck (Nwk) and belong to the PCH protein family which regulate the F-actin polymerization and regulate the interactions between cell membrane and cytoskeleton. Because hair cell stereocilia and cuticular plate are mainly composed of F-actin, we speculate that FCHSD1 and FCHSD2 could play an important role in development and maintenance of stereocilia and cuticular plate.Fchsdl and Fchsd2 cDNA sequences have been identified a few years ago, but the biological functions of FCHSD1 and FCHSD2 in mammals are still unclear. It is necessary to investigate the difference of function between FCHSD1 and FCHSD2. The study on FCHSD1 and FCHSD2 not only helps us to understand the biological functions of FCHSD1 and FCHSD2, but also helps us to further understand molecular mechanisms of development and maintenance of stereocilia and the cuticular plate, which could provide theoretical basis on clinical diagnosis and treatment of hearing loss.CONTENTS1. To detect the expression of mouse Fchsdl and Fchsd2 in different tissues.2. To detect the locations of FCHSDland FCHSD2 in cells or in mouse auditory hair cells.3. To identify the interacting proteins with FCHSD1 or FCHSD2.4. To study on the cell biological function of FCHSD1 and FCHSD2, such as the function of FCHSD1 and FCHSD2 on binding phospholipids in vitro and regulating the F-actin polymerization in vitro and so on.5. To study on the function of FCHSD1 and FCHSD2 in the hearing conduction with the knockout mice.METHODS1. Analysis the expression of Fchsdl and Fchsd2 in mouse different tissues1) The total RNA from mouse different tissues was extracted using RNeasy Micro Kis according to the manufacture’s protocol;2) RT-PCR analyzed the expression of FCHSD1 and FCHSD2 in the different tissues.2. Detection the localizations of FCHSD1 and FCHSD2 in COS7 cells1) The cDNAs encoding FCHSD1 and FCHSD2 were amplified by PCR and cloned into the pEGFP-C2;2) The constructed plasmids were then transformed into the COS7 cells;3) The proteins’localizations in the COS7 cells were detected with a confocal microscopy.3. Detection the localizations of FCHSD1 and FCHSD2 in mouse auditory hair cells1) Mouse cochlea was sectioned and the basilar membrane was stripped out;2) Whole-Mount immunostaining. The basilar membrane was incubated with antibodies;3) The localizations of FCHSD1 and FCHSD2 were detected in mouse auditory hair cells with a confocal microscopy.4. Identification the interacting proteins with FCHSD1 or FCHSD21) Utilizing the yeast two-hybrid screening, we filtered the interacting proteins with FCHSD1 or FCHSD2;2) Utilizing Co-immunoprecipitation, we verified these proteins’interaction.5. Detection the functions of FCHSD1 and FCHSD2 in binding phospholipids in vitro1) Construction expression plasmids. The cDNAs encoding FCHSD1ΔC and FCHSD2ΔC were subcloned into the pGEX-4T-2;2) E. coli BL-21 was used to express these fusion proteins with IPTG in the appropriate concentration;3) These induced proteins were purified with Sepharose 4B column;4) The phospholipids binding ability of FCHSD1 and FCHSD2 were detected in vitro with PIP strips.6. Detection the functions of FCHSD1 and FCHSD2 in regulating WASP-Arp2/3-mediated F-actin polymerization in vitro1) Construction expression plasmids. The cDNAs encoding FCHSD1ΔC, FCHSD2ΔC and hWASP145 were subcloned into the pGEX-4T-2 or pET-28a (+);2) E. coli BL-21 was used to express the fusion proteins with IPTG in the appropriate concentration;3) These induced proteins were purified with Sepharose 4B column (or Ni-NTA column) and further purified with molecular sieves or ion exchange chromatography;4) The functions of FCHSD1 and FCHSD2 in regulating WASP-Arp2/3-mediated F-actin polymerization were detected in vitro.7. Detection the proliferate rate of COS7 cells stably expressing MYC-FCHSD1 or MYC-SNX91) The COS7 cell lines stably expressing MYC-FCHSD1 or MYC-SNX9 were selected with appropriate concentration of G418;2) The proliferation of COS7 cells stably expressing MYC-FCHSD1 or MYC-SNX9 were detected with MTT method.8. Establishing the FCHSDlor FCHSD2 knockout vectors1) The long arm DNA fragments and the short arm DNA fragments of FCHSD1 and FCHSD2 were amplified from genomic DNA of mouse ES cells.;2) The long arm DNA fragments and the short arm DNA fragments were orderly constructed into the pKO-NTKV 1901 vector;3) The linearized FCHSD1or FCHSD2 knockout plasmids were transferred into mouse embryonic stem cells with the electrotransfer method;4) The ES cells with homologous recombination were screened.RESULTS1. FCHSD2 is more homology with Nervous Wreck (Nwk) than FCHSD1 Mouse FCHSDl and FCHSD2 share similar domain organization with their fly ortholog Nwk. we found that FCHSD2 is over 41% amino-acid identity with Nwk for the F-BAR domains, but FCHSD1’s F-BAR domain has only 28% identity with Nwk’s F-BAR domain. However, for SH3 domains, FCHSD1 and FCHSD2 are similar with Nwk’s. In addition, the C-terminus of FCHSD2 is longer than FCHSD1 about 60 amino acids (Fig.1-1).2. FCHSD2 is widely expressed in mouse different tissues, but FCHSD1 is detected mainly in the nervous systems1) RT-PCR analysis showed that FCHSD1 is mainly expressed in mouse spiral ganglion and cerebral cortex, but FCHSD2 is more widely expressed. FCHSD2 is detected in the organ of corti, utricle, spiral ganglion, tongue, eyes, liver, cerebellum and other tissues (Fig.1-2).2) Western blot analysis showed that mouse FCHSD1 and FCHSD2 are expressed in the organs of cochlea and vestibular (Fig.1-3).3. FCHSDl is detectable in cuticular plate but FCHSD2 is detectable in mouse stereocilia We utilized whole-mount staining to detect FCHSD1 and FCHSD2 localizations in mouse auditory hair cells. It showed that FCHSD1 immunoreactivity was mainly detected in the cuticular plat (Fig.1-4A, B), but FCHSD2 is along the whole stereociliary shaft in a punctuate pattern, where it is visible as two parallel rows flanking the F-actin core (Fig.1-4C, D).4. EGFP-FCHSDl and EGFP-FCHSD2 have similar positions in COS7 cells Figure 2-1 showed that EGFP-FCHSDl and EGFP-FCHSD2 located in the cytoplasm of COS7 cells with a scanning confocal microscopy. EGFP-FCHSD1 is a more uniform distribution in cytoplasm and the expression of EGFP-FCHSD1 is higher than EGFP-FCHSD2. EGFP-FCHSD2 is mainly around the nucleus.5. The interacting proteins with FCHSD1 or FCHSD2 are different1) Yeast two-hybrid experiment showed that the interacting proteins with FCHSD1, containing SNX9, ITSN1, ITSN2 and so on (Fig.2-3). Because FCHSD2 is self-action and cannot use the yeast two-hybrid system to screen its interacting proteins.2) Western blot results showed that FCHSD1 interacts with FCHSD1, FCHSD2 and SNX9, but FCHSD2 interacts with FCHSD1, FCHSD2, WASP, N-WASP and so on (Fig.2-2and 2-4 A).6. The F-BAR domain of FCHSD1 is co-immunoprecipitated with the SH3 domain of SNX9 According to the composition of proteins, we divided FCHSD1 into two portions and divided SNX9 into three portions (Fig.2-4B). The co-immunoprecipitation results showed that the F-BAR domain of FCHSD1 interacts with the SH3 domain of SNX9(Fig.2-4C,D).7. Mouse SNX9 is expressed in organs of cochlea and vestibular and immunore-activity colocalized with FCHSD1 in the cuticular plate1) Western blot results showed that SNX9 is expressed in mouse cochlea and vestibular (Fig.2-5).2) The whole-mount staining results showed that SNX9 is expressed in the cuticular plate of inner hair cells and colocalized with FCHSD1 in the same confocal section (Fig.2-6).8. FCHSD1 is binding the kinds of phospholipids as the same as FCHSD2 in vitro1) GST-FCHSD1ΔC, GST-FCHSD2ΔC and GST were expressed in E. coil BL-21 with IPTG and purified with Sepharose 4B column. Western blot results were showed the expression of these GST proteins in Figure 3-1A.2) Phospholipids binding assay showed that FCHSD1 and FCHSD2 are mainly binding to PtdIns(3)P, PtdIns(5)P and PS (Fig.3-2).9. The function of FCHSD1 is different from FCHSD2 in regulating WASP-Arp2/3- mediated F-actin polymerization1) GST, GST-FCHSD1ΔC, GST-FCHSD2ΔC, GST-SNX9 and His-hWASP145 were induced to express and then were purified. SDS-PAGE electrophoresis gel was used to detect the expression of these proteins (Fig.3-1B).2) FCHSD1 does not affect the F-actin polymerization, but FCHSD1 greatly enhances the stimulation of SNX9 to F-actin polymerization (Fig.3-3 A, B).3) FCHSD2 stimulates the WASP-Arp2/3 mediated F-actin polymerization, but SNX9 does not affect the regulation of FCHSD2 to F-actin polymerization (Figure 3-3).10. Stable expression of MYC-FCHSD1 or MYC-SNX9 can inhibit the proliferation of COS7 cell1) Using G418 (final concentration of 500μg/ml) to screen stably expressing MYC-FCHSD1 or MYC-SNX9 COS7 cell lines, we obtained one clone of stable expressing MYC-FCHSD1 cell lines and two cell lines of stable expression MYC-SNX9. They are named MYC-SNX9-3 and MYC-SNX9-5 (Figure 3-4).2) We used the MTT method to detect the proliferation rate of COS7 cell lines stably expressing MYC-FCHSD1 or MYC-SNX9. The results showed that, comparing to the control group, stably overexpressing MYC-FCHSD1 or MYC-SNX9 in COS7 cells could significantly inhibit the cell growth (Figure 3-5).11. Establishing the FCHSDl or FCHSD2 knockout vectors1) Constructing and identificating the FCHSDl knockout vector The long arm DNA fragment (5062bp) and the short arm DNA fragment (2082bp) of FCHSDlwere amplified from genomic DNA of mouse ES cells. These homologous recombination arm DNA fragments were cloned into the pKO-NTKV 1901 vector (Figure 4-1). After the occurrence of homologous recombination, the first exon to the sixth exon of FCHSD1 (5226bp DNA) is replaced by the DNA sequence of neomycin resistance gene Neo.2) Constructing and identificating the FCHSD2 knockout vector The long arm DNA fragment (4271bp) and the short arm DNA fragment (1941bp) of FCHSD2 were amplified from genomic DNA of mouse ES cells. These homologous recombination arm DNA fragments were cloned into the pKO-NTKV 1901 vector (Figure 4-2). After the occurrence of homologous recombination, the upstream gene sequence including the first exon of FCHSD2 (19532bp) is replaced by the DNA sequence of neomycin resistance gene Neo.COUNCLUTIONS1. FCHSD2 is more conservative than FCHSD1 compared with Nwk. Proteins interacting with Nwk, for example WASP and N-WASP, interact with FCHSD2 but not FCHSD1. It is indicating that the function of FCHSD2 is similar with Nwk.2. The FCHSD1 expression is different from FCHSD2 in mouse different tissues. Fchsdl appears to be most abundantly expressed in the cortex, whereas Fchsd2 is generally expressed at easily detectable levels.3. The FCHSD1 position is different from FCHSD2 in mouse auditory hair cells, which suggests that the function of FCHSD1 may be different from FCHSD2 in auditory hair cells.4. In vitro, FCHSD1 and FCHSD2 can binding the similar kinds of phospholipids.5. The function of FCHSD1 is different from FCHSD2 in regulating WASP-Arp2/3-mediated F-actin polymerization. FCHSD1 with the help of SNX9 indirectly controls the F-actin polymerization, but FCHSD2 immediately stimulates the F-actin polymerization.6. When COS7 cells stably express MYC-FCHSD1 or MYC-SNX9, their growth rate is reduced. |
