鞭毛相關蛋白PF20 (Paralyzed flagella protein)是存於細胞纖毛與鞭毛的蛋白質，在具有這些運動胞器生物中，它的基因在演化上呈現高度的保守性。對單細胞寄生蟲布魯氏錐蟲（Trypanosoma brucei）來說，基因TbPF20缺失，不僅移動性失能，細胞也因而無法正常進行胞質分裂，讓此基因和錐蟲存活有決定性的關係。TbPF20共有589個胺基酸，經過序列比對預測出SMC (structure maintenance of chromosome) domain與WD-repeats domain兩個高保守性的功能區，而在SMC domain之前的序列與其他物種的PF20比較下呈現低相似度，稱為變異區。先前的研究發現含有SMC domain及WD-repeats domain的截短化TbPF20片段對哺乳動物上皮細胞造成細胞週期停滯在G2/M期，成對中心體（centrosome）無法分離到細胞兩極，不能順利進行細胞分裂而產生細胞毒殺效應。另外，SMC片段刪除的TbPF20（簡稱SacII）及單獨WD-repeats片段（WDN）也造成細胞週期停滯在G2/M期，中心體無法分離，甚至細胞移動性降低以及微管系統出現捲曲、束狀及被剪切的不正常現象。特別的是，許多SacII蛋白在細胞中的分布大多形成斑點狀聚集在細胞的周圍邊緣。為了探討各個片段毒殺細胞的貢獻與原因，以及驗證先前在影像實驗上的發現，本研究將TbPF20截成不同的重要片段，接到GST蛋白形成融合蛋白，並且利用原核表現系統與親和純化法得到各片段蛋白，而其中發現SMC刪除片段（GST-Sac）、WD-repeats（GST-WD）蛋白會進入到包含體（inclusion body）內，必須以尿素純化法純化。接著將這些純化的融合蛋白再做為餌，進行GST pull down 實驗，以西方點墨法分析與餌交互作用的細胞內蛋白。實驗結果顯示與GST控制組比較， GST-Sac、GST-WD及WD前含SMC片段（GST-272）三種融合蛋白能與某些細胞內的蛋白質交互作用，且GST-Sac能夠結合微管切解蛋白劍蛋白p60次單元，驗證影像實驗中與SacII共存在的結果。此外，實驗結果也顯示Sac、WD及272蛋白會結合γ-tubulin及β-actin，其它片段則無，且所有片段都不結合微管蛋白，證明此GST pull down實驗所釣到的交互作用蛋白具有專一性。或許因TbPF20結合了細胞骨架相關蛋白及中心體相關蛋白影響有絲分裂而毒殺細胞，然而更詳細的機制以及其它交互作用的蛋白則需再進一步研究。

PF20 (Paralyzed flagella protein) is an essential and conserved gene of organism with motile cilium/flagellum. For protozoan Trypanosoma brucei , a parasite living in mammalian bloodsteam, TbPF20 is essential for cytokinesis in addition to motility. Within 589 residues, bioinformatic method indicated TbPF20 containing SMC (structure maintenance of chromosome) domain and WD-repeats domain. Previous studies showed that truncated TbPF20 bearing SMC and WD-repeats domains confered cytotoxicity in mammalian epithelial cells due to G2/M arrest and centrosome disjunction failure. Furthermore, SMC domain deleted TbPF20 (SacII) construct and construct bearing WD repeat domain only (WDN) both not only conferred cytotoxicity due to G2/M arrest and centrosome disjunction failure but also reduced cell motility. These cells also showed abnormal microtubule patterns. Interestingly, SacII cells displayed delicate puncta, thin thread-like beads along cell edges. To delineate contribution of each domain on the cytotoxicity, and to confirm the discovery of image data, various deletion mutants of TbPF20 were tagged with GST protein, expressed in prokaryotic expression system. Two constructs, SMC deleted fragment (GST-Sac) and WD-repeats only fragment (GST-WD), were harvested as inclusion bodies and renatured back with urea. Through GST affinity method these TbPF20 segments as baits were used to fish proteins from MDCK lysate. The results reported that GST-Sac, GST-WD and pre-WD SMC containing fragment (GST-272) would bind some proteins in MDCK cells. Western blot revealed that katanin p60, a microtubule-severing enzyme, could be pulled down only through SacII bait confirming the image data that SacII colocalized with katanin p60. Moreover, GST-Sac, GST-WD and GST-272 also specificly bound γ-tubulin and β-actin, but not bound β-tubulin. The possibility of cytotoxicity of TbPF20 in blocking cell mitosis may due to interaction with cytoskeleton related proteins and centrosome related proteins. The detail mechanisms and other possible interacted proteins of TbPF20 await further investigation.

1. Grab, D.J. & Kennedy, P.G. Traversal of human and animal trypanosomes across the blood-brain barrier. J Neurovirol 14, 344-351 (2008).
2. Lundkvist, G.B., Kristensson, K. & Bentivoglio, M. Why trypanosomes cause sleeping sickness. Physiology (Bethesda) 19, 198-206 (2004).
3. Field, M.C. & Carrington, M. The trypanosome flagellar pocket. Nat Rev Microbiol 7, 775-786 (2009).
4. Absalon, S., et al. Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei. J Cell Sci 121, 3704-3716 (2008).
5. Dutcher, S.K. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet 11, 398-404 (1995).
6. Hildebrandt, F., Benzing, T. & Katsanis, N. Ciliopathies. N Engl J Med 364, 1533-1543 (2011).
7. Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat Rev Mol Cell Biol 8, 880-893 (2007).
8. Broadhead, R., et al. Flagellar motility is required for the viability of the bloodstream trypanosome. Nature 440, 224-227 (2006).
9. Kohl, L., Robinson, D. & Bastin, P. Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes. EMBO J 22, 5336-5346 (2003).
10. Lodish, H.F. Molecular cell biology, (W.H. Freeman and Co., New York, 2012).
11. Badano, J.L., Mitsuma, N., Beales, P.L. & Katsanis, N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet 7, 125-148 (2006).
12. Smith, E.F. & Lefebvre, P.A. PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella. Mol Biol Cell 8, 455-467 (1997).
13. Zhang, Z., et al. Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis. Proc Natl Acad Sci U S A 101, 12946-12951 (2004).
14. Zhang, Z., et al. Deficiency of SPAG16L causes male infertility associated with impaired sperm motility. Biol Reprod 74, 751-759 (2006).
15. Nagarkatti-Gude, D.R., et al. Spag16, an axonemal central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression. PLoS One 6, e20625 (2011).
16. Horowitz, E., et al. Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins. Mol Hum Reprod 11, 307-317 (2005).
17. Branche, C., et al. Conserved and specific functions of axoneme components in trypanosome motility. J Cell Sci 119, 3443-3455 (2006).
18. Ralston, K.S., Lerner, A.G., Diener, D.R. & Hill, K.L. Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system. Eukaryot Cell 5, 696-711 (2006).
19. Absalon, S., et al. Basal body positioning is controlled by flagellum formation in Trypanosoma brucei. PLoS One 2, e437 (2007).
20. Stirnimann, C.U., Petsalaki, E., Russell, R.B. & Muller, C.W. WD40 proteins propel cellular networks. Trends Biochem Sci 35, 565-574 (2010).
21. Zhang, Z., et al. Phosphorylation of mouse sperm axoneme central apparatus protein SPAG16L by a testis-specific kinase, TSSK2. Biol Reprod 79, 75-83 (2008).
22. Neer, E.J., Schmidt, C.J., Nambudripad, R. & Smith, T.F. The ancient regulatory-protein family of WD-repeat proteins. Nature 371, 297-300 (1994).
23. Garcia-Higuera, I., et al. Folding of proteins with WD-repeats: comparison of six members of the WD-repeat superfamily to the G protein beta subunit. Biochemistry 35, 13985-13994 (1996).
24. Neer, E.J. G proteins: critical control points for transmembrane signals. Protein Sci 3, 3-14 (1994).
25. Fong, H.K., et al. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A 83, 2162-2166 (1986).
26. Grigorieva, C. & S, S. Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiology Letters 13, 367-370 (1982).
27. Janda, L., Tichy, P., Spizek, J. & Petricek, M. A deduced Thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains. J Bacteriol 178, 1487-1489 (1996).
28. Nasmyth, K. & Haering, C.H. The structure and function of SMC and kleisin complexes. Annu Rev Biochem 74, 595-648 (2005).
29. Landschulz, W.H., Johnson, P.F. & McKnight, S.L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-1764 (1988).
30. Kerppola, T.K. & Curran, T. The transcription activation domains of Fos and Jun induce DNA bending through electrostatic interactions. EMBO J 16, 2907-2916 (1997).
31. Hirano, T. At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7, 311-322 (2006).
32. Watanabe, Y. The importance of being Smc5/6. Nat Cell Biol 7, 329-331 (2005).
33. Bloom, K. & Joglekar, A. Towards building a chromosome segregation machine. Nature 463, 446-456 (2010).
34. Niki, H., Jaffe, A., Imamura, R., Ogura, T. & Hiraga, S. The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. EMBO J 10, 183-193 (1991).
35. Roll-Mecak, A. & McNally, F.J. Microtubule-severing enzymes. Curr Opin Cell Biol 22, 96-103 (2010).
36. Zhang, D., et al. Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration. Nat Cell Biol 13, 361-370 (2011).
37. Seebeck, T., Schneider, A., Kung, V., Schlaeppi, K. & Hemphill, A. The Cytoskeleton of Trypanosoma brucei - the Beauty of Simplicity. Protoplasma 145, 188-194 (1988).
38. Kohl, L. & Gull, K. Molecular architecture of the trypanosome cytoskeleton. Mol Biochem Parasitol 93, 1-9 (1998).
39. Gull, K. Host-parasite interactions and trypanosome morphogenesis: a flagellar pocketful of goodies. Curr Opin Microbiol 6, 365-370 (2003).
40. DuBois, K.N., et al. NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions. PLoS Biol 10, e1001287 (2012).
41. Hill, K.L. Parasites in motion: flagellum-driven cell motility in African trypanosomes. Curr Opin Microbiol 13, 459-465 (2010).
42. Nogales, E. Structural insights into microtubule function. Annu Rev Biochem 69, 277-302 (2000).
43. Brinkley, B.R. Microtubule organizing centers. Annu Rev Cell Biol 1, 145-172 (1985).
44. Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature 312, 237-242 (1984).
45. Janosi, I.M., Chretien, D. & Flyvbjerg, H. Structural microtubule cap: stability, catastrophe, rescue, and third state. Biophys J 83, 1317-1330 (2002).
46. Margolin, G., et al. The mechanisms of microtubule catastrophe and rescue: implications from analysis of a dimer-scale computational model. Mol Biol Cell 23, 642-656 (2012).
47. Janke, C. & Bulinski, J.C. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat Rev Mol Cell Biol 12, 773-786 (2011).
48. Gull, K. The cytoskeleton of trypanosomatid parasites. Annu Rev Microbiol 53, 629-655 (1999).
49. Sharp, D.J. & Ross, J.L. Microtubule-severing enzymes at the cutting edge. J Cell Sci 125, 2561-2569 (2012).
50. Vale, R.D. Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts. Cell 64, 827-839 (1991).
51. McNally, F.J. & Vale, R.D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419-429 (1993).
52. Vale, R.D. AAA proteins. Lords of the ring. J Cell Biol 150, F13-19 (2000).
53. Buster, D., McNally, K. & McNally, F.J. Katanin inhibition prevents the redistribution of gamma-tubulin at mitosis. J Cell Sci 115, 1083-1092 (2002).
54. Hazan, J., et al. Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nat Genet 23, 296-303 (1999).
55. Errico, A., Claudiani, P., D'Addio, M. & Rugarli, E.I. Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Hum Mol Genet 13, 2121-2132 (2004).
56. Sanderson, C.M., et al. Spastin and atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners. Hum Mol Genet 15, 307-318 (2006).
57. Grüneberg, H. Two new mutant genes in the house mouse. J. Genet. 45, 22-28 (1943).
58. Truslove, G.M. The anatomy and development of the fidget mouse. J. Genet. 54, 64-86 (1956).
59. Yang, Y., Mahaffey, C.L., Berube, N. & Frankel, W.N. Interaction between fidgetin and protein kinase A-anchoring protein AKAP95 is critical for palatogenesis in the mouse. J Biol Chem 281, 22352-22359 (2006).
60. Zhang, D., Rogers, G.C., Buster, D.W. & Sharp, D.J. Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes. J Cell Biol 177, 231-242 (2007).
61. Hartman, J.J., et al. Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell 93, 277-287 (1998).
62. Hartman, J.J. & Vale, R.D. Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286, 782-785 (1999).
63. Roll-Mecak, A. & Vale, R.D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363-367 (2008).
64. Cassimeris, L. Cell division: eg'ing on microtubule flux. Curr Biol 14, R1000-1002 (2004).
65. McNally, K., Audhya, A., Oegema, K. & McNally, F.J. Katanin controls mitotic and meiotic spindle length. J Cell Biol 175, 881-891 (2006).
66. Srayko, M., O'Toole E, T., Hyman, A.A. & Muller-Reichert, T. Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis. Curr Biol 16, 1944-1949 (2006).
67. Roll-Mecak, A. & Vale, R.D. Making more microtubules by severing: a common theme of noncentrosomal microtubule arrays? J Cell Biol 175, 849-851 (2006).
68. Dymek, E.E., Lefebvre, P.A. & Smith, E.F. PF15p is the chlamydomonas homologue of the Katanin p80 subunit and is required for assembly of flagellar central microtubules. Eukaryot Cell 3, 870-879 (2004).
69. Sharma, N., et al. Katanin regulates dynamics of microtubules and biogenesis of motile cilia. J Cell Biol 178, 1065-1079 (2007).
70. Dymek, E.E. & Smith, E.F. PF19 encodes the p60 catalytic subunit of katanin and is required for assembly of the flagellar central apparatus in Chlamydomonas. J Cell Sci 125, 3357-3366 (2012).
71. Casanova, M., et al. Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids. Mol Microbiol 71, 1353-1370 (2009).
72. Mennella, V., et al. Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nat Cell Biol 7, 235-245 (2005).
73. Fritz-Laylin, L.K., et al. The genome of Naegleria gruberi illuminates early eukaryotic versatility. Cell 140, 631-642 (2010).
74. Fritz-Laylin, L.K. & Cande, W.Z. Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation. J Cell Sci 123, 4024-4031 (2010).
75. Hames, R.S., et al. Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells. Exp Cell Res 314, 574-589 (2008).
76. Oku, T., et al. Two regions responsible for the actin binding of p57, a mammalian coronin family actin-binding protein. Biol Pharm Bull 26, 409-416 (2003).
77. Benz, C., Clucas, C., Mottram, J.C. & Hammarton, T.C. Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin. PLoS One 7, e30367 (2012).
78. Fry, A.M. The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 21, 6184-6194 (2002).
79. Hames, R.S. & Fry, A.M. Alternative splice variants of the human centrosome kinase Nek2 exhibit distinct patterns of expression in mitosis. Biochem J 361, 77-85 (2002).
80. Mardin, B.R., et al. Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat Cell Biol 12, 1166-1176 (2010).
81. Lamberti, A., et al. Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics. Biochimie 93, 1738-1746 (2011).
82. Silina, K., et al. Sperm-associated antigens as targets for cancer immunotherapy: expression pattern and humoral immune response in cancer patients. J Immunother 34, 28-44 (2011).