臺灣博碩士論文加值系統

形態發生為發育過程中，為因應組織及器官上之需要，而產生的細胞形狀、細胞極性及細胞數目的改變。目前，我們對形態發育的分子機制並不瞭解，過去的研究顯示，決定細胞極性的基因與細胞骨架蛋白在形態發生的過程中扮演很重要的角色。因此我們使用果蠅眼睛桿小體的形態發生作為模式，來探討細胞骨架蛋白Glued對桿小體形態發生的作用。桿小體為具有感光功能的微絨毛構造，聚集於感光細胞的頂部，感光細胞的頂部為一平滑膜構造，發育過程中微絨毛構造會聚集在頂部並形成桿小體。Glued蛋白為dynactin complex中一個次單位，與Arp1及其他許多次單位共同組成dynactin 複合體。過去研究顯示，dynactin可與cytoplasmic dynein作用而參與許多細胞內運輸及細胞骨架重組的功能。桿小體發育過程中，感光細胞會發生形狀改變，並有許多胞內運輸的作用，因此推論dynactin可能參與桿小體的形態發生。
本研究發現在30~40% 蛹期之間，阻礙Glued基因的正常功能會導致桿小體發育的異常，而在40% 蛹期以後Glued對桿小體的形態發生則沒有明顯的作用。因此，我們認為Glued對桿小體的發育有顯著的影響，不過這個影響只發生於特定的發育階段。此外，我們製作Arp1基因的基因轉殖果蠅，利用熱處理啟動子誘導轉殖Arp1基因的過度表現，藉此干擾正常Arp1基因的功能。實驗結果顯示過度表現轉殖Arp1基因對正常Arp1基因的功能並無影響，我們推測由於轉殖基因包含完整長度的Arp1基因，因此過度表現轉殖Arp1基因不會對正常Arp1基因的功能造成影響。

Morphogenesis is a developmental process that involves in change of cell shape or number of cell resulting in formation of a specific tissue or organ. Previous studies have shown that the cell polarity genes and cytoskeletons play a critical role in morphogenesis. However, the mechanism to regulate morphogenesis remains largely unknown. We used rhabdomere morphogenesis in Drosophila as a model to study the role of microtubule motor protein, Glued in mediating morphogenesis. Rhabdomere is photosensitive microvilli that localized on the apical surface of the photoreceptor. During early development, the photoreceptor apex is a smooth membrane structure. Gradually, the microvilli will be deposited on the apical surface and form the rhabdomere. During rhabdomere morphogenesis, massive vesicle transport and rearrangement of cytoskeletons occurs in photoreceptors. Cytoplasmic dynein is shown to participate in mediating vesicle transport. Thus, Glued, the largest subunit of dynactin complex may interact with cytoplasmic dynein to regulate rhabdomere morphogenesis.
In this research, we found Glued plays a critical role in mediating rhabdomere morphogenesis. Altering Glued’s activity by overexpression dominant negative Glued in transgenic fly disrupts normal rhabdomere morphogenesis at 30% to 40% of pupal development (pd). After 40% pd., Glued shows no significant effects in rhabdomere formation. In addition, transgenic flies that overexpress Arp1 showed no obviously eye phenotype suggesting overexpressed Arp1 may be not able to titrate the normal Arp1’s function.

中文摘要………………………………………………………………………1
Abstract………………………………………………………………………2
前言……………………………………………………………………………3
一、形態發生…………………………………………………………………3
二、果蠅眼睛發育模式………………………………………………………4
三、Glued的研究……………………………………………………………6
四、Arp1的研究……………………………………………………………9
材料與方法…………………………………………………………………11
一、果蠅品系………………………………………………………………11
二、蛹期發育判斷及37℃熱誘導反應……………………………………11
三、穿透式電子顯微鏡法…………………………………………………12
四、Arp1基因轉殖果蠅的製作與觀察……………………………………13
結果…………………………………………………………………………17
一、正常果蠅w1118眼睛桿小體的發育…………………………………17
二、過度表現C端缺失之Glued基因對桿小體的影響……………………18
三、Arp1基因轉殖果蠅的製作與觀察……………………………………19
討論…………………………………………………………………………21
一、Glued在桿小體形態發生扮演的功能………………………………21
二、Arp1基因轉殖果蠅的觀察……………………………………………23
參考文獻……………………………………………………………………26
圖目…………………………………………………………………………32
個人資料……………………………………………………………………50

Allan, V. (2000). Dynactin. Curr. Biol. 10(12), R432.
Allen, M. J., Shan, X., Caruccio, P., Froggett, S. J., Moffat, K. G., and Murphey, R. K. (1999). Targeted expression of truncated glued disrupts giant fiber synapse formation in Drosophila. J. Neurosci. 19(21), 9374-9384.
Berleth, T., Burri, M., Thoma, G., Bopp, D., Richstein, S., Frigerio, G., Noll, M., and Nusslein-Volhard, C. (1988). The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7, 1749-1756.
Bingham, J. B., and Schroer, T. A. (1999). Self-regulated polymerization of the actin-related protein Arp1. Curr. Biol. 9 (4), 223-226.
Bixby, J. L., Grunwald, G. B., and Bookman, R. J. (1994). Ca++ influx and neurite growth in response to purified N-cadherin and laminin. J. Cell Biol. 127, 1461-1475.
Boylan, K., Serr, M., and Hays, T. (2000). A molecular genetic analysis of the interaction between the cytoplasmic dynein intermediate chain and the glued (dynactin) complex. Mol. Biol. Cell 11(11), 3791-3803.
Burkhardt, J. K., Echeverri, C. J., Nilsson, T, and Vallee, R.B. (1997). Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J. Cell Biol. 139(2), 469-84.
Chang, H. Y., and Ready, D. F. (2000). Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science 290(5498), 1978-1980.
Clark, I. B., and Meyer, D. I. (1999). Overexpression of normal and mutant Arp1α (centractin) differentially affects microtubules organization during mitosis and interphase. J. Cell Sci. 112, 3507-3518.
Cole, N. B., and Lippincott-Schwartz, J. (1995). Organization of organelles and membrane traffic by microtubules. Curr. Opin. Cell Biol. 7, 55-64.
Eaton, S., and Simons, K. (1995). Apical, basal, and lateral cues for epithelial polarization. Cell 82(1), 5-8.
Echeverri, C. J., Paschal, B. M., Vaughan, K. T., and Vallee, R. B. (1996). Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132, 617-633.
Fan, S. -S., and Ready, D. F. (1997). Glued participates in distinct microtubule-based activities in Drosophila eye development. Development 124, 1497-1507.
Farshori, P., and Holzbaur, E. L. F. (1997). Dynactin phosphorylation is modulated in response to cellular factors. Biochem. Biophys. Res. Commun. 232, 810-816.
Fyrberg, C., Ryan, L., Kenton, M., and Fyrberg, E. (1994). Genes encoding actin-related proteins of Drosophila melanogaster. J. Mol. Biol. 241, 498-503.
Garces, J. A., Clark, I. B., Meyer, D. I., and Vallee, R. B. (1999). Interaction of the p62 subunit of dynactin with Arp1 and the cortical actin cytoskeleton. Curr. Biol. 9 (24), 1497- 1500.
Gill, S. R., Schroer, T. A., Szilak, I., Steuer, E. R., Sheetz, M. P., and Cleveland, D. W. (1991). Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J. Cell Biol. 115(6), 1639-1650.
Habermann, A., Schroer, T. A., Griffiths, G., and Burkhardt, J. K. (2001). Immuno-localization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles. J. Cell Sci. 114, 229-240.
Harris, E. A., Stark, W. S., and Walker, J. A. (1976). Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J. Physiol. (Lond.) 256, 415-439.
Harte, P. J., and Kankel, D. R. (1982). Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster. Genetics 101, 477-501.
Holleran, E. A., Tokito, M. K., Karki, S., and Holzbaur, E. L. F. (1996). Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles. J. Cell Biol. 135(6), 1815-1830.
Holzbaur, E. L. F., Hammarback, J. A., Paschal, B. M., Kravit, N. G., Pfister, K. K., and Vallee, R. B. (1991). Homology of a 150k cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature 351, 579-583.
Kahana, J. A., Schlenstedt, G., Evanchuk, D. M., Geiser, J. R., Hoyt, M. A., and Silver, P. A. (1998). The yeast dynactin complex is involved in partitioning the mitotic spindle between mother and daughter cells during anaphase B. Mol. Biol. Cell 9, 1741-1756.
Karki, S. K., and Holzbaur, E. L. F. (1995). Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J. Biol. Chem. 272, 5887-5891.
Karki, S. K., Tokito, M. K., and Holzbaur, E. L. F. (1997). Casein kinase II binds to and phosphorylates cytoplasmic dynein. J. Biol. Chem. 272, 5887-5891.
Kron, S. J., and Gow, N. A (1995). Budding yeast morphogenesis: signaling, cytoskeleton and cell cycle. Curr. Opin. Cell Biol. 7(6), 845-855.
Kumer, J. P., and Ready, D. F. (1995). Rhodopsin plays an essential structural role in Drosophila photoreceptor development. Development 121, 4359-4370.
Lafont, F., Burkhardt, J. K., and Simons, K. (1994). Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature 372(6508), 801-803.
Liu, J.-K., Di Persio, M. C., and Zaret, K. S. (1991). Extracellular signals that regulate liver transcription factors during hepatic differentiation in vitro. Mol. Cell Biol. 11, 773-784.
Longley, R. L., Jr., and Ready, D. F. (1995). Integrins and the development of three-dimensional structure in the Drosophila compound eye. Dev. Biol. 171, 415-433.
McGrail, M., Gepner, J., Silvanovich, A., Ludmann, S., Serr, M., and Hays, T. S. (1995). Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex. J. Cell Biol. 131(2), 411-425.
Meyerowitz, E. M. and Kankel, D. R. (1978). A genetic analysis of visual system development in Drosophila melanogaster. Dev. Biol. 62, 112-142.
Muhua, L., Karpova, T. S., and Cooper, J. A. (1994). A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration. Cell 78, 669-679.
Pfister, K. K., Benashski, S. E., Dillman, J. F. 3rd, Patel-King R. S., King, S. M. (1998). Identification and molecular characterization of the p24 dynactin light chain. Cell Motil. Cytoskeleton 41(2), 154-167.
Plamann, M., Minke, P. F., Tinsley, J. H., and Bruno, K. S. (1994). Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi. J.Cell Biol. 127(1), 139-149.
Plough, H. H. and Ives, P. T. (1935). Induction of mutations by high temperature in Drosophila. Genetics 20, 42-69.
Ready, D. F., Hanson, T. E., Benzer, S. (1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol. 53, 217-240.
Schafer, D. A., Gill, S. R., Cooper, J. A., Heuser, J. E., and Schroer, T. A. (1994). Ultrastructural analysis of the dynactin complex : an actin-related protein is a component of filament that resembles F-actin. J. Cell Biol. 126, 403-412.
Schroer, T. A., and Sheetz, M. P. (1991). Two activators of microtubule-based vesicle transport. J. Cell Biol. 115(5), 1309-1318.
Shrimankar, P. V., Schriefer, L. A., and Waterson, R. H. (1994). Actin-related proteins in C.elegans. Worm Breeders Gaz. 13, 56.
Swaroop, A., Swaroop, M., and Garen, A. (1987). Sequence analysis of the complete cDNA and encoded polypeptide for the Glued gene of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 84, 6501-6505.
Tamkun, J. W., DeSimone, D. W., Fonda, D., Patel, R. S., Buck, C., Horwitz, A. F., and Hynes, R. O. (1986). Structure of integrin, a glycoprotein involved in transmembrane linkage between fibronectin and actin. Cell 46, 271-282.
Tepass, U., Theres, C., and Knust, E. (1990). crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61(5), 787-799.
Tinsley, J. H., Minke, P. F., Bruno, K. S., and Plamann, M. (1996). p150Glued, the largest subunit of the dynactin complex , is nonessential in Neurospora but required for nuclear distribution. Mol. Biol. Cell 7, 731-742.
Tzanakakis, E. S., Hansen, L. K., Hu, W. S. (2001). The role of actin filaments and microtubules in hepatocyte spheroid self-assembly. Cell Motil. Cytoskeleton 48(3), 175-189.
Vaughan, K. T., and Vallee, R. B. (1995). Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J. Cell Biol. 131, 1507-1516.
Viamontes, G. I., Fochtmann, L. J., and Kirk, D. L. (1979). Morphogenesis in Volvox: analysis of critical variables. Cell 17(3), 537-550.
Waterman-Storer, C. M., Karki, S., and Holzbaur, E. L. F. (1995). The p150Glued component of the dynactin complex binds to both the microtubules and the actin-related protein centractin (Arp-1). Proc. Natl. Acad. Sci. USA 92, 1634-1638.
Wolff, T. and Ready, D. F. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster, A. M. Arias and M. Bate, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 1277-1325.
Wu, Q., Sandrock, T. M., Turgeon, B. G., Yoder, O. C., Wirsel, S. G., Aist, J. R. (1998). A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis. Mol Biol. Cell 9(1), 89-101.
Yap, A. S., Manley, S. W. (2001). Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid. Cell Motil. Cytoskeleton 48(3), 201-212.