Figure 1.
The conserved protein Kank directly interacts with EB1 in vitro and requires EB1 to localise to microtubule ends in cultured S2 cells.
(A) Drosophila Kank contains the conserved structural elements of Kank family proteins. Drosophila Kank also contains putative, conserved motifs including nuclear localisation signals (NLS in green), nuclear export signals (NES in grey) and SxIP/EB1 binding motifs (in purple). (B) Kank produced by in vitro transcription/translation in the presence of 35S-methionine was successfully pulled down by bacterially expressed MBP-EB1 but not by MBP alone. Autoradiograph and protein staining are shown at the top and bottom panels respectively. The input is equivalent to 30% of the pull downs. (C) Cultured S2 cells were transfected with GFP-Kank and immunostained for GFP and α-tubulin. The GFP signal localises to the cytoplasm, largely to microtubule ends (yellow arrows). Minor localisation was also observed at the cell periphery. (D) S2 cells transfected with GFP-Kank were immunostained for GFP and EB1. The GFP signal largely co-localised with EB1 comets (yellow arrows). (E) Immunoblotting confirmed the reduction of EB1 by RNAi. (F) Kank requires EB1 to localise to microtubule ends. RNAi of EB1 in S2 cells led to delocalisation of GFP-Kank from microtubule ends, compared to control β-lactamase RNAi cells. The percentage of cells with GFP-Kank signal localised to the majority of visible EB1 comets (>50%, estimate) was counted. The GFP-Kank signal at EB1 comets was significantly reduced in EB1 RNAi cells compared to control RNAi cells and control cells without RNAi. Error bars show the standard error of the mean. For all microscopy images yellow boxes are areas magnified in images shown below and co-localisation is indicated by yellow arrows. Scale bars = 5 µm.
Figure 2.
Kank/EB1 binding is via an EB1 binding, SxIP, motif in the middle region of Kank.
(A) A summary of Kank truncations and their microtubule plus end localisation. ++ (strong localisation), + (weak localisation), - (no localisation). (B) Kank truncations which contain the middle region of Kank can co-localise with EB1 in S2 cells. S2 cells transfected with GFP-fused truncations were co-stained for GFP, EB1 and DNA. Co-localisation was observed (yellow arrows). (C) The SxIP motif in Kank was mutated to SxNK. (D) S2 cells transfected with GFP-Kank(I764N,P765K) were co-stained for GFP and EB1. Mutation of the SxIP motif in the middle region of Kank abolished co-localisation of Kank with EB1 (yellow arrows). (E) Mutation of the SxIP motif significantly decreased the percentage of cells with GFP signal localised to the majority of the observed EB1 comets. Significance was determined by Fishers exact chi squared test. Error bars show the standard error of the mean. For all microscopy images yellow boxes are areas magnified in images shown below and co-localisation is indicated by yellow arrows. Scale bars = 5 µm.
Figure 3.
Kank can localise transiently to the nucleus of S2 cells.
(A) Kank(1–500) and Kank(889–1224) exhibit nuclear localisation, while truncations of Kank which contain the middle region do not. S2 cells were transfected with GFP-fused Kank truncations. Cells were then co-stained for GFP, α-tubulin and DNA. (B) Kank shuttles between the nucleus and the cytoplasm in some cells. S2 cells transfected with GFP-Kank were incubated with media containing leptomycin B for 3–3.5 hours, to inhibit nuclear export. Control cells were incubated with media containing methanol, the solvent for leptomycin B. Nuclear localisation was observed more frequently in leptomycin B treated cells than control cells. Significance was determined by Fishers exact chi squared test. Error bars show the 95% confidence interval. (C) A summary of Kank truncations and their nuclear localisation. + and – indicate the presence and absence of the nulcear localisation with (+LB) or without (–LB) Leptomycin B. ND (Not done). Scale bars = 5 µm.
Figure 4.
Kank is expressed throughout development but is dispensable for viability and fertility.
(A) kank (CG10249) is a ∼27 kb gene found at 51D2 on chromosome arm 2R. Putative isoforms are shown. Those in black are likely to be expressed while those in grey are less likely to be expressed based on ModEncode data. The cDNA clone GH03482 that we used in our analysis represents isoform A of kank (highlighted in blue). This isoform lacks the KN motif found in other Kank proteins (shown in purple). (B) Kank was deleted using transposons containing FRT sites. Firstly, the appropriate two transposons flanking the Kank coding sequence were introduced in trans positions on homologous chromosomes (i). A flippase was induced to promote recombination between the FRT sites (ii) and generated a deletion of the intervening sequence (iii) (C) The fragment of Kank(489–900) used for generating an antibody against the Kank protein. (D–G) The Kank antibody detected the endogenous protein in all lifecycle stages examined by immunoblotting in wild type but not in Kank deletion mutants. Kank was detected in embryos 21–24 hrs after egg laying (D), in 3rd instar larvae (E), in male and female late pupae [ = L] and early pupae of undetermined gender [ = E] (F), and in both male and female adult flies (G).
Figure 5.
Kank localises to muscle-tendon attachment sites in late stage Drosophila embryos.
(A) The ∼160 kDa Kank band was detected in embryos from 3–6 hours after egg laying (AEL). The amount of Kank detected by immunoblotting was observed to increase during embryonic development. The ∼140 kDa band becomes apparent 15–18 hours AEL. (B) An antibody against Kank(489–900) stained a distinct pattern in stage 16/17 embryos. This staining was not observed in kank deletion mutants. The 22c10 antibody, which highlights neurons, was used to orient embryos. (C) β3-tubulin staining reveals the structure of microtubules in somatic muscle cells. Co-staining with the Kank antibody showed that Kank localises at sites of muscle attachment to the epidermis. (D) A schematic of Drosophila embryonic somatic musculature with sites of Kank staining indicated. Scale bars = 25 µm.
Figure 6.
Deletion of kank did not affect muscle cell morphology or microtubule organisation.
(A) Closer examination of the lateral transverse muscle shows Kank staining near sites where microtubule ends are attached to the periphery of muscle cells. (B) No clear differences were seen in the overall organisation of somatic musculature, or in the organisation of their microtubules, between wild type and kankΔ as imaged with β3-tubulin staining. Scale bars = 25 µm.