Identification of a Novel Microtubule-Binding Protein in Giardia lamblia
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.
Abstract
Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.
참고문헌 (19)
- Elmendorf HG, "The cytoskeleton of Giardia lamblia", Int J Parasitol, 33, pp.3~28 (2003)
- Desai A, "Microtubule polymerization dynamics", Annu Rev Cell Dev Biol, 13, pp.83~117 (1997)
- Akhmanova A, "Tracking the ends: a dynamic protein network controls the fate of microtubule tips", Nat Rev Mol Cell Biol, 9, pp.309~322 (2008)
- Dawson SC, "Kinesin-13 regulates flagellar, interphase, mitotic microtubule dynamics in Giardia intestinalis", Eukaryot Cell, 6, pp.2354~2364 (2007)
- Kim J, "Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein", PLoS One, 9, pp.e97850~ (2014)
- Kim J, "Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules", Parasitol Int, 57, pp.465~471 (2008)
- Holberton DV, "Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular-weight protein associated with microribbon structures", J Cell Sci, 47, pp.39~166 (1981)
- Lourenço D, "Proteomic analysis of the ventral disc of Giardia lamblia", BMC Res Notes, 5, pp.41~ (2012)
- Hagen KD, "Novel structural components of the ventral disc and lateral crest in Giardia intestinalis", PLoS Negl Trop Dis, 5, pp.e1442~ (2011)
- Woessner DJ, "The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment", Eukaryot Cell, 11, pp.292~301 (2012)
- Keister DB, "Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile", Trans R Soc Trop Med Hyg, 77, pp.487~488 (1983)
- Harlow E, "Antibodies: A Laboratory Manual, Immunization", pp.53~63 (1988), Cold Spring Harbor Laboratory
- Singer SM, "Episomal and integrated maintenance of foreign DNA in Giardia lamblia", Mol Biochem Parasitol, 92, pp.59~69 (1998)
- Kim J, "Comparative proteomic analysis of trophozoites versus cysts of Giardia lamblia", Parasitol Res, 104, pp.475~479 (2009)
- Gourguechon S, "Rapid tagging and integration of genes in Giardia intestinalis", Eukaryot Cell, 10, pp.142~145 (2011)
- Debec A, "Centrioles: active players or passengers during mitosis?", Cell Mol Life Sci, 67, pp.21173~22194 (2010)
- Crossley R, "Immunocytochemical differentiation of microtubules in the cytoskeleton of Giardia lamblia using monoclonal antibodies to γ-tubulin and polyclonal antibodies to associated low molecular weight proteins", J Cell Sci, 80, pp.233~252 (1986)
- Nohynkova E, "Localization of gamma-tubuin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis", Eur J Cell Biol, 79, pp.438~445 (2000)
- Lauwaet T, "Mining the Giardia genome and proteome for conserved and unique basal body proteins", Int J Parasitol, 41, pp.1079~1092 (2011)