Roles of end‐binding 1 protein and gamma‐tubulin small complex in cytokinesis and flagella formation of Giardia lamblia

Abstract

Giardia lamblia is a unicellular organism with two nuclei, a median body, eight flagella, and an adhesive disk. γ‐Tubulin is a microtubule (MT)‐nucleating protein that functions in the γ‐tubulin small complex (γ‐TuSC) in budding yeast. In this study, G. lamblia γ‐tubulin (Glγ‐tubulin) was found to bind to another MT‐binding protein, namely G. lamblia end‐binding protein 1 (GlEB1), via both in vivo and in vitro assays. Hemagglutinin (HA)‐tagged Glγ‐tubulin localized to the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. The knockdown of Glγ‐tubulin expression using an anti‐Glγ‐tubulin morpholino resulted in a decreased growth rate and an increased failed cytokinesis cells of Giardia . The formation of median bodies was affected, and the central pair of MTs in flagella was frequently missing in the Giardia treated with an anti‐Glγ‐tubulin morpholino. G. lamblia γ‐tubulin complex protein 2 (GlGCP2) and GlGCP3, which are putative components of γ‐TuSC, were co‐immunoprecipitated with HA‐tagged Glγ‐tubulin in Giardia extracts. The knockdown of GlGCP2 and GlGCP3 expression also resulted in decreased formation of both the median body and flagella MTs. Knockdown of Glγ‐tubulin, GlGCP2, and GlGCP3 expression affected localization of GlEB1 in G. lamblia . In addition, decreased level of GlEB1 caused reduced formation of median body and the central pair of flagella MTs. These results indicated that Glγ‐tubulin plays a role in MT nucleation for median body formation and flagella biogenesis as a component of Glγ‐TuSC in Giardia and GlEB1 may be involved in this process.

Nek8445, a protein kinase required for microtubule regulation and cytokinesis inGiardia lamblia

“…, 2014 ). Intriguingly, EB1 knockdown in Giardia has been shown to reduce median body volume, flagella length, and cause loss of axoneme central pairs ( Kim and Park, 2019 ). Although less severe, these phenotypes are similar to what we observe in Nek8445-depleted cells.…”

A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

“…Localization of GlPLK at basal bodies, which function as an MTOC, indicated that GlPLK might play a role in MT nucleation. A previous study demonstrated that depletion of the γ-tubulin ring complex (γ-TuSC) affects MT nucleation, resulting in shortening of the agella [17]. Overexpression of dominantnegative mutant kinesin-13, a motor protein, produced Giardia with longer agella and defective mitotic spindles [40].…”

“…Expression of HAtagged GlPLK was con rmed by western blotting. Giardia trophozoites carrying pΔ.pac [17] were included as a control.…”

“…Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, following which immunoreactive proteins were visualized using an Enhanced Chemiluminescence System (Thermo Fisher Scienti c, Waltham, MA, USA). Membranes were incubated in a stripping buffer (Thermo Fisher Scienti c) at room temperature for 20 min and then reacted with polyclonal rat antibodies against protein disul de isomerase 1 (PDI1; GL50803_29487) of G. lamblia (1:10,000) as the loading control [17].…”

“…In contrast, the extracts of G. lamblia carrying the vector control, pΔ.pac, did not produce any immunoreactive bands in the same analysis. Western blotting of the same membrane with anti-GlPDI1 antibodies [17] served as a loading control for the total amount of protein in the extracts used for this assay.…”