Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis

Abstract

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.

Figure 1. Kebab interacts with EB1.

Cell extract from a stable cell line expressing Kebab-GFP was incubated with bacterially produced MBP and MBP-EB1. MBP and MBP-EB1 were pulled down and subjected to western blot using an anti-Kebab antibody (the upper panel). One twentieth of the cell extract was run, relative to the pull-down fractions. Protein staining of the same membrane is shown in the lower panel. Kebab-GFP is specifically pulled down with EB1.

Figure 2. Kebab localises to kinetochores and spindle microtubules.

(A) Kebab localises to mitotic kinetochores. S2 cells were transfected with a plasmid expressing Kebab-GFP under the actin promotor, and immunostained for GFP, Cid and DNA. The arrowheads indicate the position of centromeres. Bar = 10 µm. (B) Kebab foci were located outside of CID foci. Magnified images of sister centromeres. Bar = 1 µm. (C) Kebab localises to kinetochores in anaphase. S2 cells expressing Kebab-GFP were immunostained for α-tubulin, GFP and DNA. Bar = 10 µm. (D) Kebab localises to spindle microtubules in telophase. Bar = 10 µm. (E) S2 cells expressing Kebab-GFP were incubated with colchicine and immunostained. In the inserts, the boxed region containing a single chromosome was magnified. The arrowheads indicate the position of kinetochores at the primary constriction. Kinetochore signals were greatly reduced. Bar = 10 µm. (F) S2 cells expressing Kebab-GFP were incubated with paclitaxel and immunostained. The arrowheads indicate the position of kinetochores. Bar = 10 µm.

Figure 3. Kebab localisation dynamically change during mitosis.

S2 cells stably expressing Kebab-GFP and mCherry-α-tubulin were observed under a spinning disc confocal microscope. Bars = 10 µm. (A) Still images of Kebab-GFP localisation at different stages of mitosis are indicated on the left side of B. (B) A kymograph of maximum intensity projection with the long axis of the spindle (X-axis) against time (Y-axis). Arrowhead indicates accumulating kinetochore signals during anaphase. (C) A diagram of the kymograph in B indicating cellular location of signals. (D) Increase of Kebab signal intensity on kinetochores during anaphase. Kebab signal intensities on kinetochores above the background were plotted from late metaphase to the end of anaphase for 5 random cells. The intensity values were normalised against the maximal value in each anaphase.

Figure 4. Kebab has EB1 binding motifs, an atypical CH domain and a coiled-coil region.

(A) A diagram of the Kebab protein structure. (B) Similarity between the CH domains of human Ndc80 (hNdc80), D. melanogaster Ndc80 (dNdc80) and D. melanogaster Kebab (dKebab). The identical residues between two proteins were shown in bold blue letters. The asterisks indicate the residues important for the microtubule binding in human Ndc80. (C) A series of truncations and mutations tested for their localisation. KC and MT indicates the degree of localisation to kinetochores and microtubules. (++) full localisation, (+) weak localisation, (±) a trace of localisation, (−) no localisation. (D) EB1 binding assay of Kebab with mutated SxIP motifs. Radiolabelled proteins were in vitro translated and mixed with beads coupled with MBP and MBP-EB1. Pull-down fractions were run along with the original input (25% of pull-down fractions) and radiolabelled Kebab was detected by autoradiograph. Specific EB1 binding activity by this assay is indicated together with kinetochore or microtubule localisation (KC or MT). (E) Kinetochore localisation of a full-length GFP-Kebab and Kebab with both EB1 binding motifs mutated (ΔIPs). (F) Kinetochore localisation in EB1 depleted cells. Bar = 10 µm.

Figure 5. Kebab is dispensable for mitotic progression and fly viability and fertility.

(A) Mitotic index of S2 cells without or after colchicine incubation. S2 cells were subjected to RNAi of kebab and a control. S2 cells were immunostained for phospho-H3 at Serine 10 and DNA. The mitotic index was the proportion of phospho-H3 positive cells. Bars indicate standard deviations. (B) Time from nuclear envelope breakdown to anaphase onset. S2 cells expressing mCherry-RCC1 were subjected to RNAi of kebab and a control, and observed in live cells. (C) The genomic region around kebab. The open boxes, grey boxes and kinked lines indicate non-coding exons, coding exons and introns, respectively. The triangle indicates the position of the P-element insertion in k09932. The parentheses indicate regions deleted in each keb mutant.