The role of the Kinesin-13 family protein TbKif13-2 in flagellar length control of Trypanosoma brucei

Abstract

TbKif13-2, a member of the microtubule-depolymerising Kinesin-13 family was localised at the tip of the flagellum in Trypanosoma brucei. Its predicted activity suggested a role in the regulation of axonemal length. However, using gene deletion and overexpression of TbKif13-2 we show that, in procyclic T. brucei, this kinesin has only a very limited effect on flagellar length. Gene deletion resulted in no significant elongation of the flagellum and overexpression only slightly decreased flagellar length and the rate of growth of a new flagellum during cell division. This is in contrast to studies in Leishmania major, where overexpression of the TbKif13-2 homologue resulted in a significant length reduction of the flagellum. Knockout of TbKif13-2 has, however, an effect on the initial growth of the emerging new flagellum. In conclusion, we show that TbKif13-2 has only a marginal impact on flagellar length in T. brucei.

Graphical abstract

Fig. 1

TbKif13-2 localisation and the effect of TbKif13-2 knockout in procyclic T. brucei cells. (A) Immunofluorescence of TbKif13-2myc induced cells, fixed with 3.7% (w/v) formaldehyde in PBS. Fixed cells were permeabilised with 0.1% (v/v) NP40 and labelled with anti-myc antibody and stained with DAPI. Cells with a single (1K1N) and a mature and new (2K1N) flagellum are shown. Bars, 2 μm. (B) RT-PCR from cDNA made from total RNA isolated from (1) Control PC449 cells (2) TbKif13-2 knockout cells. The upper panel shows the absence of TbKif13-2 transcript in knockout cells and the lower panel represents a control PCR reaction using the cohesin subunit SMC3 transcript. (C) Trypanosome cytoskeletons labelled with L8C4 which stains the PFR. Cytoskeletons were prepared by extracting whole trypanosome cells with 0.1% (v/v) NP40 before fixation with 3.7% (w/v) formaldehyde in PBS. Bar, 2 μm. (D) Histogram of flagellar length distribution in WT PC449 cells, TBKif13-2 knockout (−/−) cells and TbKif13-2myc induced cells. Only mature flagella in 1N1K cells were scored. (E) Lines of best fits illustrating the increase in the growing new flagellum in relation to the distance between dividing kinetoplasts. Only cells where the two kinetoplasts can be easily be resolved were measured. The plot reveals that there is no change in the rate of flagellar outgrowth between PC449 cells and TbKif13-2 knockout cells but TbKif13-2myc expression results in a decrease in the rate of flagellum outgrowth. Additional information is in the Supplementary data files.