研究生: |
龐威 Wei-Pang |
---|---|
論文名稱: |
斑馬魚仔魚側線毛細胞上的TRP通道之研究 The functional study of transient receptor potential (TRP) ion channels in lateral-line hair cells of zebrafish larvae |
指導教授: |
林豊益
Lin, Li-Yih |
學位類別: |
碩士 Master |
系所名稱: |
生命科學系 Department of Life Science |
論文出版年: | 2011 |
畢業學年度: | 99 |
語文別: | 中文 |
論文頁數: | 61 |
中文關鍵詞: | 斑馬魚胚胎 、毛細胞 、TRP通道 、機械性感覺 |
英文關鍵詞: | zebrafish larva, hair cells, TRP channels, mechanosensory |
論文種類: | 學術論文 |
相關次數: | 點閱:156 下載:4 |
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魚類的側線系統是由毛細胞(hair cells)和支持細胞組成的機械性感覺器官,負責感覺外在水體的流動。毛細胞的纖毛束在受到機械性刺激時會開啟非選擇性的陽離子通道,對於鈣離子有較高的選擇性。流入細胞的鈣離子會引發胞內的訊息傳導,使毛細胞傳出感覺訊號。
然而脊椎動物內耳與魚類側線毛細胞上的機械性感覺通道的分子組成仍具爭議性,目前主要認為transient receptor potential (TRP) channels家族的蛋白較可能是參與的分子,但證據仍有所欠缺。本研究利用斑馬魚胚胎為模式動物,進一步研究側線毛細胞上參與機械性感覺的分子。我們利用掃描式離子選擇性電極技術scanning ion selective electrode technique (SIET)在活體胚胎毛細胞上測得鈣離子流入,並證實這個通道會被neomycin、gentamicin、La3+及BAPTA所抑制。利用morphlino gene knockdown技術將TRPA1、TRPN1及TRPV4表現弱化,發現TRPA1及TRPN1弱化後毛細胞上鈣離子流入顯著降低。免疫染色也顯示這三個蛋白質在纖毛上表現,並且在基因弱化後,表現量明顯下降。本研究結果顯示,斑馬魚側線毛細胞上的TRPA1與TRPN1通道扮演著機械性感覺的角色。
The lateral-line, a mechanosensory organ of fish, is composed of hair cells and supporting cells. The mechanosensory channel on the hair bundles of hair cells is a non-selective cation channel with higher Ca2+ permeability. Mechanical deflection of the sensory hair bundles causes Ca2+ influx via the channel and further induces sensory transduction. Recently, a group of Ca2+ channels, the TRP (transient receptor potential) channels, was suggested to be the mechanosensory channel on hair cells. However, the molecular identity of the channel is still controversial. In this study, we used zebrafish as an in vivo model to investigate the mechanosensory channels on the lateral-line hair cell of newly hatch larvae. We used a non-invasive Scanning Ion-Selective Electrode Technique (SIET) to measure the Ca2+ influx at the hair bundles as the hair were depressed by the Ca2+-sensitive microprobe. We successfully detect Ca2+ influx with this new method and found that the Ca2+ influx was blocked by neomycin, gentamicin, La3+ and BAPTA which were shown to block the mechanosensory channel in other studies. Isoform- specific antibody were generated to localize 3 zebrafish TRP proteins (TRPA1a, TRPN1 and TRPV4) on the lateral line hair bundles. Using morpholino-gene knockdown technique, the protein expression of the TRP channels were blocked and leaded to a remarkable decline of Ca2+ influx, suggesting that the TRP channels are involved in the mechanosensory of zebrafish lateral-line hair cells.
Assad, J. A., Shepherd, G. M. G., and Corey, D. P. (1991). Tip-link
Integrity and Mechanical Transduction in Vertebrate Hair Cells. Neuron. 7:985-994.
Clapham, D. E. (2003). TRP channels as cellular sensors. Nature.
426:517-524.
Chaudière, C. D., Sapède, D., Soubiran, F., Decorde, K., Gompel, N.,
Ghysen, A. (2003). The lateral line of zebrafish: a model system for the analysis of morphogenesis and neural development in vertebrates. Biol Cell. 95:579–587.
Corey, D. P. and Hudspeth, A. J. (1979). Ionic basis of the receptor
potential in a vertebrate hair cell. Nature. 281:675-677.
Corey, D. P., overos, J. G., Holt, J. R., Kwan, K. Y., Lin, S. Y.,
Vollrath, M. A., Amalfitano, A., Cheung, E. L-M., Derfler, B. H., Duggan, A., Geleoc, G. S. G., Gray, P. A., Hoffman, M. P., Rehm, H. L., Tamasauskas, D. & Zhang, D. S. (2004). TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature. 432.
Corey, D. P. (2006). What is the hair cell transduction channel. J Physiol.
576.1.
Coffin, A. B., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E.
W. (2009). Extracellular divalent cations modulate aminoglycoside-induced hair cell death in the zebrafish lateral line. Hear Res. 253:42–51.
Cuajungco, M. P., Grimm, C., Heller, S. (2007). TRP channels as candidates for hearing and balance abnormalities in vertebrates. Biochim Biophys Acta. 1022–1027.
Dijkgraaf, S. (1989). A short personal review of the history of lateral line
research. In: Coombs, S., Görner, P., Münz, H. (Eds.), The
Mechanosensory Lateral Line. Neurobiology and Evolution.
Springer-Verlag, New-York, pp. 7–14.
Farris H. E., LeBlanc, C. L., Goswami, J. and Ricci, A. J. (2004).
Probing the pore of the auditory hair cell mechanotransducer channel
in turtle. J Physiol. 558:769-792.
Forge, A., and Schacht, J. (2000). Aminoglycoside Antibiotics.
Audiol Neurootol. 5:3-22.
Gale, J. E., Marcotti, W., Kennedy, H. J., Kros, C. J., and
Richardson, G. P. (2001). FM1-43 Dye Behaves as a Permeant Blocker of the Hair-Cell Mechanotransducer Channel. J. Neuroscl. 21(18):7013–7025.
Ghysen, A. and Chaudière, C. D. (2007). The lateral line microcosmos.
Genes Dev. 21:2118–2130.
Ghysen, A. and Chaudière, C. D. (2004). Development of the zebrafish
lateral line. Curr Opin Neurobiol. 14:67–73.
Greene, C.C., McMillan, P. M., Barker, S. E., Kurnool, P., Lomax,
M. I., Burmeister, M., Lesperance, M. M. (2001). DFNA25, a novel locus for dominant non-syndromic hereditary hearing impairment, maps to 12q21-24. Am J Hum Genet. 8:254–260.
Guler, A. D., Lee, H., Iida, T., Shimizu, I., Tominaga, M., Caterina,
M. (2002). Heat-evoked activation of the ion channel, TRPV4. J. Neurosci. 22:6408–6414.
Harris, J. A., Cheng, A. G., Cunningham, L. L., Macdonald,G.,
Raible, D. W and Rubel, E. W. (2003). Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio rerio). J Assoc Res Otolarynqol. 04: 219–234.
Hamill, O.P. and McBride, D.W. Jr. (1996). The pharmacology of
mechanogated membrane ion channels. Pharmacol Rev.
48: 231–252.
Kim, J., Chung, Y.D., Park, D. Y., Choi, S. Shin, D.W., Soh, H.,
Lee, H.W., Son, W., Yim, J., Park, C. S., Kernan, M.J., Kim, C. (2003). A TRPV family ion channel required for hearing in Drosophila. Nature. 424:81–84.
Kroese, A. B. A., Das, A., and Hudspeth, A. J. (1989). Blockage of the
transduction channels of hair cells in the bullfrog’s sacculus by aminoglycoside antibiotics. Hear Res. 37:203-218.
Kwan, K. Y., Allchorne, A. J., Vollrath, M. A., Christensen, A. P.,
Zhang, D. S., Woolf, C. J. and Corey, D. P. (2006). TRPA1 Contributes to Cold, Mechanical, and Chemical Nociception but Is Not Essential for Hair-Cell Transduction. Neuron. 50:277–289.
Latorre, R. and Editor, G. (2009). Perspectives on TRP Channel
Structure and the TRPA1 Puzzle. J Gen Physiol. 133:227–229.
Liedtke, W., Choe, Y., Marti-Renom, M. A., Bell, A. M., Denis, C. S.,
Sali, A., Hudspeth, A.J., Friedman, J.M., Heller, S. (2000). Vanilloid receptor related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell. 103: 525–535.
Lumpkin, E. A. and Michael, J. C. (2007). Mechanisms of sensory
transduction in the skin. Nature. 445:858-865.
Mangos, S., Liu, Y., and Drummond, I. A. (2007). Dynamic expression
of the osmosensory channel trpv4 in multiple developing organs in
zebrafish. Gene Expr Patterns. 7:480–484.
Mirjam, F., Anja, L., Ksenia, J. G., Stephan, C. F. N., Helmut, S.,
Rik, I. L. E. (2009). Zebrafish (Danio rerio) neuromast: Promising
biological endpoint linking developmental and toxicological studies.
Aquat Toxicol. 307–319.
Mutai, H. and Heller, S. (2003). Vertebrate and invertebrate TRPV-like
mechanoreceptors. Cell Calcium. 33:471–478.
Nagata, K., Duggan, A., Kumar, G., and Garcı´a-Anoveros, J. (2005).
Nociceptor and Hair Cell Transducer Properties of TRPA1, a
Channel for Pain and Hearing. J Neurosci. 25(16 ): 4052– 4061.
Nilius, B. and Voets, T. (2005). TRP channels: a TR(I)P through a world
of multifunctional cation channels. Pflugers Arch. 451:1–10.
Ohmori, H. (1985). Mechano-electrical transduction currents in isolated
vestibular hair cells of the chick. J Physiol. 359:189-217.
Ou, H. C., Raible, D. W., and Rubel, E. W. (2007). Cisplatin-induced
hair cell loss in zebrafish (Danio rerio) lateral line. Hear Res.
233:46–53.
Ou, H. C., Santos, F., Raible, D W., Simon, J. A., and Rubel, E. W.
(2010). Drug screening for hearing loss: Using the zebrafish lateral
line to screen for drugs that prevent and cause hearing loss. Drug
Discov Today. 15.
Owens, K. N., Coffin, A. B., Hong, L. S., Bennett, K. O. C., Rubel, E.
W., Raible, D. W. (2009). Response of mechanosensory hair cells of
the zebrafish lateral line to aminoglycosides reveals distinct cell
death pathways. Hear Res. 253:32–41.
Owens, K. N., Cunningham, D., Macdonald, G., Rubel, E. W., Raible,
D. W. and Pujol, R. (2007). Ultrastructural Analysis of
Aminoglycoside-Induced Hair Cell Death in the Zebrafish Lateral
Line Reveals an Early Mitochondrial Response. J. Comp. Neurol.
502:522–543
Prober, D. A., Zimmerman, S., Myers, B. R., McDermott Jr, B. M.,
Kim, S. H., Caron, S., Rihel, J., Krezel, L. S., Julius, D.,
Hudspeth, A. J. and Schier, A. F. (2008). Zebrafish TRPA1
Channels Are Required for Chemosensation But Not for
Thermosensation or Mechanosensory Hair Cell Function. J. Neuro sci. 28(40):10102–10110.
Ricci, A. J. and Fettiplace, R. (1998). Calcium permeation of the turtle
hair cell mechanotransducer channel and its relation to the
composition of endolymph. J. Physiol. 506:159-173.
Shin, J. B., Adams, D., Paukert, M., Siba, M., Sidi, S., Levin, M.,
Gillespie, P. G., and Grunder, S. (2005). Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells. Proc Natl Acad Sci U S A. 102:12572–12577.
Sidi, S., Friedrich, R. W. and Nicolson, T. (2003). NompC TRP
Channel Required for Vertebrate Sensory Hair Cell Mechanotransduction. Science. 301.
Siemens,J., Lillo, C., Dumont, R. A., Reynolds, A., Williams, D. s.,
Gillespie, P. G., amd Müller, U. (2004). Cadherin 23 is a component of the tip link in hair-cell stereocilia. Nature. 428:950–955.
Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G. T.D.
(2000). Plant, OTRPC4, a non-selective cation channel that confers sensitivity to extracellular osmolarity. Nat. Cell Biol. 2: 695-702.
Suzuki, M., Mizuno, A., Kodaira, K., Imai, M. (2003). Impaired
pressure sensation in mice lacking TRPV4. J Biol Chem. 278: 22664–22668.
Trump, W. J. V., Coombs, S., Duncan, K., McHenry, M. J. (2010).
Gentamicin is ototoxic to all hair cells in the fish lateral line system. Hear Res.261:42–50.
Williams, J.A. and Holder, N. (2000). Cell turnover in neuromasts of zebrafish larvae. Hear Res. 143:171–181.
Yang, X. C. and Sachs, F. (1989). Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science. 243:1068–1071.