Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly

Abstract

During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.

Fig. 1

GAK is required for efficient chromosome alignment. a Hela cells were transfected with six different siRNAs targeting GAK or with an siRNA targeting GAPDH as a control. Twenty-four hours after transfection cells were re-transfected and 48 h after the first transfection cells were fixed, DNA was stained with DAPI, and the percentage of mitotic cells with misaligned chromosomes was scored in each condition. b Hela cells were transfected as in (a) with indicated siRNAs, and 48 h after the first transfection cells were lysed and GAK protein levels were determined by western blot. c, d Hela (c) or U2OS cells (d) were treated as in (a), and the average number of misaligned chromosomes was determined (c) or the percentage of mitotic cells with misaligned chromosomes was scored (d). e Hela cells were treated as in (a), but 48 h after transfection cells were fixed and stained for CLIP-170 and p150glued to visualize unattached kinetochores and DAPI was used to stain the DNA. All graphs are averages of three independent experiments with 50 cells scored per condition per experiment. Error bars represent standard deviations. Scale bar in (e) indicates 5 µm

Fig. 2

GAK is required for correct spindle assembly. a U2OS cells were transfected with indicated siRNAs. Twenty-four hours after transfection cells were re-transfected and 48 h after the first transfection cells were fixed so that spindles and DNA could be visualized by anti-α-tubulin staining and DAPI, respectively. b Cells were treated as in (a), and the spindle length was measured using the α-tubulin staining. Graph in (b) shows averages of three independent experiments with 25 cells scored per condition per experiment. Error bars represent standard deviations. Scale bar in (a) indicates 5 µm

Fig. 3

GAK is required for the generation of chromatin-derived microtubules. a–c U2OS cells were transfected with indicated siRNAs. Twenty-four hours after transfection, cells were re-transfected, and 48 h after the first transfection, cells were treated with nocodazole for 6 h, washed four times with medium to remove nocodazole, and fixed after 3 min (a, upper panels and b, c) or after 10 min (a, lower panels) and stained for α-tubulin and γ-tubulin. DAPI was used to visualize the DNA. a Representative images. b, c Quantifications. d Cells were transfected as in (a–c) but were fixed after 48 h without nocodazole treatment. Cells were then stained for HURP and α-tubulin, and DNA was stained with DAPI. HURP localizes specifically to DNA-proximal microtubules both in control cells and in GAK-depleted cells. Graphs in (b, c) show averages of three independent experiments with 25 cells scored per condition per experiment. Error bars represent standard deviations. Scale bars in (a, d) indicate 5 µm

Fig. 4

GAK mediates clathrin recruitment to the spindle. a Hela cells were transfected with indicated myc-tagged plasmids and 24 h later, cells were transfected with GAK siRNA where indicated. Forty-eight hours after siRNA transfection cells were fixed and stained for myc. DAPI was used to visualize the DNA. Chromosome alignment was then analyzed in mitotic cells expressing the indicated plasmids. b, c U2OS cells stably expressing green fluorescent protein (GFP)-clathrin light chain were transfected with indicated siRNAs. Twenty-four hours after transfection cells were re-transfected and 48 h after the first transfection cells were fixed and stained for α-tubulin and GFP. DAPI was used to visualize the DNA. a Representative cells. b Quantifications. d–g U2OS cells were transfected with indicated siRNAs. Twenty-four hours after transfection cells were re-transfected and 60 h after the first transfection cells were treated with nocodazole for 6 h, washed four times to remove nocodazole, fixed after 3 min, and stained for α-tubulin and γ-tubulin. DAPI was used to visualize the DNA. The amount of microtubules around centrosomes (visualized by γ-tubulin staining; d) or chromosomes (visualized by DAPI staining; e) was then analyzed. Images in (f) show a control cell (upper panel) and a clathrin heavy chain-depleted cell with a strong phenotype (lower panel). Graphs in (b–e, g) show averages of three independent experiments with 25 cells scored per condition per experiment in (c–e, g) and 50 cells per condition in (c). Error bars represent standard deviations. Scale bars in (a, f) indicate 5 µm