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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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Localization of GlEB1 in G. lamblia during encystation.Giardia trophozoites and encysting cells were prepared at various time-points of post-induction of encystation. (A) Fixed cells were serially reacted with mouse anti-HA antibodies (1∶100) and AlexaFluor 488-conjugated anti-mouse IgG (1∶200). (B) As a control for encystation, a separate set of trophozoites and encysting cells was reacted with anti-GlCWP1 antibodies (1∶100). The scale bar is 2 µm.
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pone-0097850-g003: Localization of GlEB1 in G. lamblia during encystation.Giardia trophozoites and encysting cells were prepared at various time-points of post-induction of encystation. (A) Fixed cells were serially reacted with mouse anti-HA antibodies (1∶100) and AlexaFluor 488-conjugated anti-mouse IgG (1∶200). (B) As a control for encystation, a separate set of trophozoites and encysting cells was reacted with anti-GlCWP1 antibodies (1∶100). The scale bar is 2 µm.

Mentions: The intracellular level of GlEB1 was found to be constitutive during encystation [13]. In this study, we examined the possibility that localization of EB1 was altered during encystation by IFA of Giardia expressing HA-tagged GlEB1 with anti-HA antibodies (Figure 3A). Up to 24 h-after induction to encystation, GlEB1 was mainly found at the nuclear membrane in the most of the Giardia cells, whereas some of the cells showed fluorescent labeling of the median bodies. In cysts at 48 h induction, fluorescence was absent in the nuclear membranes and dispersed in the cytoplasm of the cells. As a control for encystation process, intracellular location of CWP1 was also examined in trophozoites as well as encysting cells (Figure 3B). CWP1 was barely detected in trophozoites, and later found in encystation-specific vesicles in the encysting cells. Localization of CWP1 in the cyst walls was distinct in the cysts at 48 h-after induction of encystation. These results showed that the location of GlEB1 at the nuclear membrane and median bodies was changed into a condensed pattern in the cytoplasm of cysts.


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)

Localization of GlEB1 in G. lamblia during encystation.Giardia trophozoites and encysting cells were prepared at various time-points of post-induction of encystation. (A) Fixed cells were serially reacted with mouse anti-HA antibodies (1∶100) and AlexaFluor 488-conjugated anti-mouse IgG (1∶200). (B) As a control for encystation, a separate set of trophozoites and encysting cells was reacted with anti-GlCWP1 antibodies (1∶100). The scale bar is 2 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020936&req=5

pone-0097850-g003: Localization of GlEB1 in G. lamblia during encystation.Giardia trophozoites and encysting cells were prepared at various time-points of post-induction of encystation. (A) Fixed cells were serially reacted with mouse anti-HA antibodies (1∶100) and AlexaFluor 488-conjugated anti-mouse IgG (1∶200). (B) As a control for encystation, a separate set of trophozoites and encysting cells was reacted with anti-GlCWP1 antibodies (1∶100). The scale bar is 2 µm.
Mentions: The intracellular level of GlEB1 was found to be constitutive during encystation [13]. In this study, we examined the possibility that localization of EB1 was altered during encystation by IFA of Giardia expressing HA-tagged GlEB1 with anti-HA antibodies (Figure 3A). Up to 24 h-after induction to encystation, GlEB1 was mainly found at the nuclear membrane in the most of the Giardia cells, whereas some of the cells showed fluorescent labeling of the median bodies. In cysts at 48 h induction, fluorescence was absent in the nuclear membranes and dispersed in the cytoplasm of the cells. As a control for encystation process, intracellular location of CWP1 was also examined in trophozoites as well as encysting cells (Figure 3B). CWP1 was barely detected in trophozoites, and later found in encystation-specific vesicles in the encysting cells. Localization of CWP1 in the cyst walls was distinct in the cysts at 48 h-after induction of encystation. These results showed that the location of GlEB1 at the nuclear membrane and median bodies was changed into a condensed pattern in the cytoplasm of cysts.

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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