Journal of Otolaryngology and Ophthalmology of Shandong University ›› 2019, Vol. 33 ›› Issue (5): 40-43.doi: 10.6040/j.issn.1673-3770.0.2019.063

• Original Article • Previous Articles     Next Articles

Preliminary study on screening cochlear proteins interacting with myosin VI

Qingxiu YAO,Hui WANG,Zhuangzhuang LI,Dongzhen YU(),Shankai YIN   

  1. Department of Otolaryngology Head and Neck Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233，China
  • Received:2019-02-15 Revised:2019-04-16 Online:2019-09-20 Published:2019-10-15
  • Contact: Dongzhen YU E-mail:drdzyu@126.com

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Abstract: Objective

To screen for cochlear proteins that interact with myosin VI.

Method

Cochlear proteins from SD rats were extracted for co-immunoprecipitation and mass spectrometry to screen for proteins interacting with myosin VI, which were further verified by immunofluorescence of whole mount basilar membrane and cryostat sections of cochleae.

 Result

Combined immunoprecipitation and mass spectrometry yielded more than 20 proteins interacting with myosin VI, and immunofluorescence showed that spectrin αII is primarily located at the cuticular plate and the lateral wall while myosin VI is mainly located in the cell body and cuticular plate. Both myosin VI and spectrin αII were co-localized in the cuticular plate.

 Conclusion

More than 20 cochlear proteins could interact with myosin VI, suggesting that myosin VI may interact with a variety of proteins to maintain stereociliary stability.

Key words: Spectrin αII, Myosin VI, Actin, Cuticular plate

CLC Number: 

  • R764.43

Fig. 1

SDS-PAGE silver stained strip after co-immunoprecipitation"

Table 1

The results of mass spectrometry"

编号 蛋白名称 蛋白质量 肽数 GI数
1 肌球蛋白-9样蛋白 227526.00 40 392349606
血影蛋白β链 274326.94 7 61557085
2 血影蛋白α链 285216.69 4 31543764
肌动蛋白 42051.85 4 4501885
3 热休克蛋白 83572.27 7 91234898
肌动蛋白 42051.85 4 4501885
4 血影蛋白α链 285216.69 4 31543764
肌动蛋白 42051.85 4 4501885
5 血影蛋白α链 285216.69 4 31543764
肌动蛋白 42051.85 4 4501885
6 微管蛋白α-1B链 50803.87 6 34740335
肌动蛋白 42051.85 4 4501885
7 微管蛋白α-1B链 50803.87 6 34740335
微管蛋白β-5链 50095.16 5 7106439
8 微管蛋白α-1B链 50803.87 6 34740335
血影蛋白α链 285216.69 4 31543764

Fig.2

The localization of myosin VI and spectrin αII"

1 Melchionda S , Ahituv N , Bisceglia L , et al . MYO6, the human homologue of the gene responsible for deafness in Snell′s waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss[J]. Am J Hum Genet, 2001, 69(3): 635-640. doi:10.1086/323156 .
doi: 10.1086/323156
2 Ahmed ZM , Morell RJ , Riazuddin S , et al . Mutations of MYO6 are associated with recessive deafness, DFNB37[J]. Am J Hum Genet, 2003, 72(5): 1315-1322. doi:10.1086/375122 .
doi: 10.1086/375122
3 Avraham KB , Hasson T , Steel KP , et al . The mouse Snell′s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells[J]. Nat Genet, 1995, 11(4): 369-375. doi:10. 1038/ng1295-369 .
doi: 10. 1038/ng1295-369
4 Williams LH , Miller KA , Dahl HH , et al . Characterization of a novel ENU-generated myosin VI mutant mouse strain with congenital deafness and vestibular dysfunction[J]. Hear Res, 2013, 299: 53-62. doi:10.1016/j.heares. 2013.02.006 .
doi: 10.1016/j.heares. 2013.02.006
5 Hertzano R , Shalit E , Rzadzinska AK , et al . A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells[J]. PLoS Genet, 2008, 4(10): e1000207. doi:10.1371/journal.pgen. 1000207 .
doi: 10.1371/journal.pgen. 1000207
6 Ménétrey J , Bahloul A , Wells AL , et al . The structure of the myosin VI motor reveals the mechanism of directionality reversal[J]. Nature, 2005, 435(7043): 779-785. doi:10.1038/nature03592 .
doi: 10.1038/nature03592
7 Bahloul A , Chevreux G , Wells AL , et al . The unique insert in myosin VI is a structural calcium-calmodulin binding site[J]. Proceedings of the National Academy of Sciences, 2004, 101(14): 4787-4792. doi:10.1073/pnas. 0306892101 .
doi: 10.1073/pnas. 0306892101
8 Seiler C , Ben-David O , Sidi S , et al . Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish[J]. Dev Biol, 2004, 272(2): 328-338. doi:10.1016/j.ydbio.2004.05.004 .
doi: 10.1016/j.ydbio.2004.05.004
9 Self T , Sobe T , Copeland NG , et al . Role of myosin VI in the differentiation of cochlear hair cells[J]. Dev Biol, 1999, 214(2): 331-341. doi:10.1006/dbio.1999.9424 .
doi: 10.1006/dbio.1999.9424
10 Mochizuki E , Okumura K , Ishikawa M , et al . Phenotypic and expression analysis of a novel spontaneous myosin VI null mutant mouse[J]. Exp Anim, 2010, 59(1): 57-71.doi:10.1538/expanim.59.57
doi: 10.1538/expanim.59.57
11 郭纯 . 免疫共沉淀技术的研究进展[J]. 中医药导报, 2007, 13(12): 86-89. doi:10.3969/j.issn.1672-951X.2007. 12.041 .
doi: 10.3969/j.issn.1672-951X.2007.12.041
GUO Chun . Study progress of co-immunoprecipitation technology[J]. Guiding Journal of Tcm, 2007, 13(12):86-89. doi:10.3969/j.issn.1672-951X.2007.12.041 .
doi: 10.3969/j.issn.1672-951X.2007.12.041
12 Zhu M , Yang T , Wei S , et al . Mutations in the γ-actin gene (ACTG1) are associated with dominant progressive deafness (DFNA20/26)[J]. The American Journal of Human Genetics, 2003, 73(5): 1082-1091. doi:10. 1086/379286 .
doi: 10. 1086/379286
13 Belyantseva IA , Perrin BJ , Sonnemann KJ , et al . Gamma-actin is required for cytoskeletal maintenance but not development[J]. Proc Natl Acad Sci USA, 2009, 106(24): 9703-9708. doi:10.1073/pnas.0900221106 .
doi: 10.1073/pnas.0900221106
14 Rivière JB , van Bon BWM , Hoischen A , et al . De novo mutations in the actin genes ACTB and ACTG1 cause Baraitser-Winter syndrome[J]. Nat Genet, 2012, 44(4): 440-444. doi:10.1038/ng.1091 .
doi: 10.1038/ng.1091
15 Seki Y , Miyasaka Y , Suzuki S , et al . A novel splice site mutation of myosin VI in mice leads to stereociliary fusion caused by disruption of actin networks in the apical region of inner ear hair cells[J]. PLoS One, 2017, 12(8): e0183477. doi:10.1371/journal.pone.0183477 .
doi: 10.1371/journal.pone.0183477
16 Sakurai K , Hirata M , Yamaguchi H , et al . Phospholipase Cδ3 is a novel binding partner of myosin VI and functions as anchoring of myosin VI on plasma membrane[J]. Advances in Enzyme Regulation, 2011, 51(1): 171-181. doi:10.1016/j.advenzreg.2010.09.014 .
doi: 10.1016/j.advenzreg.2010.09.014
17 Salles FT , Andrade LR , Tanda S , et al . CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI[J]. Cytoskeleton (Hoboken), 2014, 71(1): 61-78. doi:10.1002/cm.21159 .
doi: 10.1002/cm.21159
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