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.
Highlights
Microtubules (MTs) are dynamic polymers involved in mitosis, organelle biogenesis, and intracellular transport [1]
An E. coli-G. lamblia shuttle plasmid was constructed to contain the DNA fragment encoding the promoter and open reading frame (ORF) of G. lamblia EB1 (GlEB1), from which End-binding 1 (EB1) is expressed as a fused form in frame with the HA epitope (Figure 1A)
The bim1D cells carrying pRS426+ PGAL1–10 EB1-C13S with C13S mutant EB1 maintained a similar level of cells with a bud-distal nucleus (62%). These results demonstrate that the EB1 homolog of G. lamblia can function to Bim1p in the budding yeast and cysteine residue #13 of GlEB1 plays a role in this process
Summary
Microtubules (MTs) are dynamic polymers involved in mitosis, organelle biogenesis, and intracellular transport [1]. Growth and catastrophe of MTs are mediated by MT-associated proteins including plus-end tracking proteins (+TIPs) which preferentially bind to the growing ends of polymerized MTs in vivo [2]. Among the +TIPs, end-binding 1 (EB1) proteins are the main regulator of the protein complexes at the plus end, as shown in mammalian EB1 [3] and yeast EB1 homologs, Mal3p [4] and Bim1p [5]. The proteins belonging to the EB1 family are composed of an N-terminal calponin homology (CH) domain, a composite domain consisting of a a-helically coiled-coli and a unique EB1 homology region, and the C-terminal acidic tail ending with an EEY/F motif [6]. The EB1 protein is one of highly conserved proteins among in all eukaryotes, including Giardia lamblia, a protozoan belonging to the earliest diverging eukaryotic lineage [9]
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