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Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.

Kim J, Nagami S, Lee KH, Park SJ - PLoS ONE (2014)

Bottom Line: Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites.Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast.These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Medical Biology and Institute of Tropical Medicine, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.

ABSTRACT
End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

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Morpholinos-mediated knockdown of GlEB1 expression in G. lamblia.Giardia trophozoites were collected at 48 and 72 h after electroporation with water (W), mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos (A). (A) Extracts of these cells were analyzed by Western blots using anti-EB1 antibodies or anti-α-tubulin antibodies. (B) Relative expression of GlEB1 in cell extracts treated with mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos. (C) The cells were attached to glass slides coated with L-lysine in a humidified chamber. The attached cells were fixed with chilled 100% methanol at -20°C for 10 min, and permeabilized with PBS/0.5% Triton X-100 for 10 min. After a 1 h incubation in blocking buffer (PBS, 5% goat serum, and 3% BSA), the cells were reacted overnight with mouse anti-GlEB1 polyclonal antibodies (1∶100). Following three times 5 min-washes with PBS, the cells were incubated with AlexaFluor 488-conugated anti-mouse IgG (1∶200; Molecular Probes) at 37°C for 1 h. Slides were mounted with VECTASHIELD anti-fade mounting medium with DAPI. They were then observed with an Axiovert 200 fluorescent microscope.
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pone-0097850-g004: Morpholinos-mediated knockdown of GlEB1 expression in G. lamblia.Giardia trophozoites were collected at 48 and 72 h after electroporation with water (W), mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos (A). (A) Extracts of these cells were analyzed by Western blots using anti-EB1 antibodies or anti-α-tubulin antibodies. (B) Relative expression of GlEB1 in cell extracts treated with mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos. (C) The cells were attached to glass slides coated with L-lysine in a humidified chamber. The attached cells were fixed with chilled 100% methanol at -20°C for 10 min, and permeabilized with PBS/0.5% Triton X-100 for 10 min. After a 1 h incubation in blocking buffer (PBS, 5% goat serum, and 3% BSA), the cells were reacted overnight with mouse anti-GlEB1 polyclonal antibodies (1∶100). Following three times 5 min-washes with PBS, the cells were incubated with AlexaFluor 488-conugated anti-mouse IgG (1∶200; Molecular Probes) at 37°C for 1 h. Slides were mounted with VECTASHIELD anti-fade mounting medium with DAPI. They were then observed with an Axiovert 200 fluorescent microscope.

Mentions: Cell extracts prepared from these cells at 48 h and 72 h post-transfection were monitored for their intracellular levels of EB1 by Western blot using anti-GlEB1 antibodies (Figure 4A). Both cells treated with mispair anti-EB1 or anti-EB1 morpholinos showed decreased amount of GlEB1 at 48 h post-transfection to 69% and 43%, respectively (Figure 4B). Amount of GlEB1 was restored to 88% and 70% of that of cells treated with water in cells at 72 h post-transfection. Decreased level of GlEB1 was also confirmed by IFA of G. lamblia cells treated with sterile water, the mispair anti-EB1, or anti-EB1 morpholinos after 48 h (Figure 4C). GlEB1 was found in nuclear membrane and median bodies in cells treated with water or mispair anti-EB1 morpholinos. In G. lamblia transfected with anti-EB1 morpholinos, GlEB1 was barely detected in nuclear membranes. Clear staining of median bodies were observed in G. lamblia cells treated with anti-EB1 morpholinos, but at lower levels than in cells treated with water or mispair anti-EB1 morpholinos.


Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.

Kim J, Nagami S, Lee KH, Park SJ - PLoS ONE (2014)

Morpholinos-mediated knockdown of GlEB1 expression in G. lamblia.Giardia trophozoites were collected at 48 and 72 h after electroporation with water (W), mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos (A). (A) Extracts of these cells were analyzed by Western blots using anti-EB1 antibodies or anti-α-tubulin antibodies. (B) Relative expression of GlEB1 in cell extracts treated with mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos. (C) The cells were attached to glass slides coated with L-lysine in a humidified chamber. The attached cells were fixed with chilled 100% methanol at -20°C for 10 min, and permeabilized with PBS/0.5% Triton X-100 for 10 min. After a 1 h incubation in blocking buffer (PBS, 5% goat serum, and 3% BSA), the cells were reacted overnight with mouse anti-GlEB1 polyclonal antibodies (1∶100). Following three times 5 min-washes with PBS, the cells were incubated with AlexaFluor 488-conugated anti-mouse IgG (1∶200; Molecular Probes) at 37°C for 1 h. Slides were mounted with VECTASHIELD anti-fade mounting medium with DAPI. They were then observed with an Axiovert 200 fluorescent microscope.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4020936&req=5

pone-0097850-g004: Morpholinos-mediated knockdown of GlEB1 expression in G. lamblia.Giardia trophozoites were collected at 48 and 72 h after electroporation with water (W), mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos (A). (A) Extracts of these cells were analyzed by Western blots using anti-EB1 antibodies or anti-α-tubulin antibodies. (B) Relative expression of GlEB1 in cell extracts treated with mispair anti-EB1 morpholinos (M), or anti-EB1 morpholinos. (C) The cells were attached to glass slides coated with L-lysine in a humidified chamber. The attached cells were fixed with chilled 100% methanol at -20°C for 10 min, and permeabilized with PBS/0.5% Triton X-100 for 10 min. After a 1 h incubation in blocking buffer (PBS, 5% goat serum, and 3% BSA), the cells were reacted overnight with mouse anti-GlEB1 polyclonal antibodies (1∶100). Following three times 5 min-washes with PBS, the cells were incubated with AlexaFluor 488-conugated anti-mouse IgG (1∶200; Molecular Probes) at 37°C for 1 h. Slides were mounted with VECTASHIELD anti-fade mounting medium with DAPI. They were then observed with an Axiovert 200 fluorescent microscope.
Mentions: Cell extracts prepared from these cells at 48 h and 72 h post-transfection were monitored for their intracellular levels of EB1 by Western blot using anti-GlEB1 antibodies (Figure 4A). Both cells treated with mispair anti-EB1 or anti-EB1 morpholinos showed decreased amount of GlEB1 at 48 h post-transfection to 69% and 43%, respectively (Figure 4B). Amount of GlEB1 was restored to 88% and 70% of that of cells treated with water in cells at 72 h post-transfection. Decreased level of GlEB1 was also confirmed by IFA of G. lamblia cells treated with sterile water, the mispair anti-EB1, or anti-EB1 morpholinos after 48 h (Figure 4C). GlEB1 was found in nuclear membrane and median bodies in cells treated with water or mispair anti-EB1 morpholinos. In G. lamblia transfected with anti-EB1 morpholinos, GlEB1 was barely detected in nuclear membranes. Clear staining of median bodies were observed in G. lamblia cells treated with anti-EB1 morpholinos, but at lower levels than in cells treated with water or mispair anti-EB1 morpholinos.

Bottom Line: Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites.Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast.These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Medical Biology and Institute of Tropical Medicine, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.

ABSTRACT
End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

Show MeSH
Related in: MedlinePlus