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First published online 16 October 2007
doi: 10.1242/jcs.007955

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Tousled-like kinase in a microbial eukaryote regulates spindle assembly and S-phase progression by interacting with Aurora kinase and chromatin assembly factors

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2280, USA

View larger version (39K): [in this window] [in a new window]   Fig. 1. RNAi silencing of TLK1 and TLK2 in the procyclic form of T. brucei. (A) Clonal cell lines harboring the respective TLK1 and TLK2 RNAi constructs were cultivated with (+Tet) or without (–Tet) tetracycline and monitored for cell growth. The insets show the levels of mRNA, monitored by northern blot, in cells before (–) and after (+) RNAi. -Tubulin (Tub) was included as a loading control. (B) Flow cytometry analysis of DNA contents in TLK1 or TLK2 RNAi cells. Insets show the percentages of cells at G1-, S- or G2/M-phases. (C) The control and TLK1 RNAi cells after 16 and 24 hours RNAi induction were tabulated for different numbers of nuclei (N) and kinetoplasts (K). The data are presented as the mean percentage±s.e. of 200 cells counted from three independent experiments. (D) Control and TLK1 RNAi cells after 24 hours induction were stained with L1C6 antibody to monitor the nucleolus, and with L8C4 and YL1/2 antibodies to label the flagella and basal bodies, respectively, and counterstained with DAPI to show the nucleus and kinetoplast. Arrows point to the basal bodies, and arrowheads indicate the flagella. (E) Spindle structures in the control and TLK1 RNAi cells after 24 hours of induction. Cells were stained with the KMX-1 antibody for the spindle and DAPI for DNA. Percentages of cells with the spindles were determined among 200 1N2K and 2N2K cells in three separate experiments. Bars, 2 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 2. Subcellular localizations of TLK1 and TLK2 in T. brucei. (A) Cells expressing HA-tagged wild-type and kinase-dead TLK1 and TLK2 were boiled in SDS sample solution and immunoblotted with mAb against (`') HA and mAb against -tubulin. (B) Cell lysates were dialyzed and incubated with (+ PPase) or without (– PPase) lambda protein phosphatase, and blotted with mAb against HA and mAb against -tubulin. (C) HA-tagged TLK1 and TLK2 were expressed in 29-13 (WT) cells or in cells harboring the AUK1 RNAi construct (AUK1 RNAi). Lysates from un-induced (– Tet) and tetracycline-induced (+ Tet) cells were immunoblotted with mAb against HA and mAb against -tubulin. (D) Subcellular localizations of wild-type and kinase-dead TLK1 and TLK2. Cells were co-stained with KMX-1 antibody for the spindle (red) and FITC-conjugated antibody against HA for HA-tagged proteins (green) and DAPI for DNA (blue). Arrows point to the brightly stained TLK1 spots, which correspond to the positions of spindle poles. (E) Subcellular localization of TLK1-HA in the AUK1 RNAi cell. The cell was co-stained with KMX-1 and antibodies against HA and counterstained with DAPI. The arrow points to a focal point of TLK1 inside the nucleus. Bars, 2 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Domains in TLK1 and TLK2 molecules required for in vivo phosphorylation and subcellular localization. Chimeric proteins constructed by exchanging the N- or the C-termini between TLK1 and TLK2 or their kinase-dead mutants were expressed as HA-tagged proteins in T. brucei. Cells were boiled in SDS sampling solution and immunoblotted with mAb against HA for the slower-migrating band as evidence for in vivo phosphorylation. The cells were also fixed, immunostained with mAb against HA and counterstained with DAPI for localizing the HA-tagged chimeric proteins in cells (also see supplementary material Fig. S4).

View larger version (70K): [in this window] [in a new window]   Fig. 4. TLK1 interacts with AUK1 in vivo and is phosphorylated by AUK1 in vitro. (A) In vitro GST pulldown assays. (B) Yeast two-hybrid assays. (C) Co-immunoprecipitation of TLK1 with AUK1 from T. brucei cells. TLK1-HA and TLK2-HA were each expressed in T. brucei cells harboring endogenous AUK1 tagged with a PTP epitope. Cells were lysed in IP lysis buffer and immunoprecipitation was performed with a pAb against HA, followed by immunoblotting with a mAb against HA or a mAb against protein C that recognizes the PTP epitope. Reciprocally, immunoprecipitation was carried out with a mAb against protein C to bring down AUK1-PTP and then blotted with a pAb against HA and a mAb against protein C, respectively. Note that no slower-migrating protein band was observed in the TLK1-HA lane because the cells were lysed in IP lysis buffer and incubated on ice for 30 minutes before boiling in SDS sample solution. The incubation might have dephosphorylated the phosphorylated protein. (D,E) AUK1 phosphorylates TLK1 (D) and TLK2 (E) in vitro. The wild-type and kinase-dead (KD) mutants of AUK1, TLK1 and TLK2 were expressed as GST-fusion proteins in E. coli, purified and mixed in various combinations for kinase assays. (F) In vitro phosphorylation of histone H3 by the purified wild-type and kinase-dead mutant (KD) TLK1, TLK2 and AUK1 recombinant proteins.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Effect of TLK1 and AUK1 double knockdown on spindle formation and chromosome segregation in T. brucei. The effect of TLK1 and AUK1 double knockdown was compared with those from TLK1 or AUK1 single knockdowns. (A) Northern blot with AUK1, the 5'-UTR of TLK1 or -tubulin as probes. (B) Comparison of growth of TLK1 and AUK1 double knockdown cells with those of the single knockdown cells. (C) Effect of AUK1 and TLK1 double knockdown on spindle formation. The 1N2K and 2N2K cells were labeled with the KMX-1 antibody (left panel) and examined for spindle structures. The data are presented as the mean percentage±s.e. of 200 cells counted from three independent experiments (right panel). (D) Double knockdowns of TLK1 and AUK1 enriched the 1N2K cells by 10-20% in comparison with those from the single knockdowns. The data are presented as the mean percentage±s.e. of 200 cells from three separate experiments. Bar, 2 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 6. The two Asf1 homologs are required for promoting S-phase progression in T. brucei. (A) Single and double knockdowns of Asf1A and Asf1B in T. brucei. A northern blot was performed with Asf1A, Asf1B or -tubulin sequences as probes. (B) Effects of the single and double knockdowns on cell growth. (C) Effects of the single and double knockdowns on cell cycle progression. The percentages of cells at G1-, S- and G2/M-phases were determined and plotted against time post induction.

View larger version (55K): [in this window] [in a new window]   Fig. 7. TLK1, Asf1A and Asf1B act in the same pathway of promoting S-phase progression in T. brucei. (A) In vitro GST pulldown assays. (B) Co-immunoprecipitation of TLK1 with Asf1A or Asf1B from T. brucei. TLK1-Ty and TLK2-Ty were coexpressed with Asf1A-HA and Asf1B-HA in T. brucei. Immunoprecipitation was performed with a mAb against Ty (BB2) and the immunoprecipitates on western blots were immunostained with a pAb against HA and a mAb against Ty, respectively. Conversely, immunoprecipitation was performed with the pAb against HA followed by immunoblotting with the mAb against Ty and the mAb against HA, respectively. Note that no slower-migrating band was observed in the TLK1-HA lane owing to cell lysis in the IP lysis buffer for 30 minutes on ice. (C) In vitro phosphorylation of Asf1A and Asf1B by TLK1 and TLK2. Wild-type and kinase-dead (KD) mutants of TLK1 and TLK2, as well as Asf1A and Asf1B, were expressed as GST fusion proteins in E. coli, purified and used for kinase assays in the presence of [-32P]ATP. (D) Triple knockdowns of Asf1A, Asf1B and TLK1 in T. brucei. A northern blot was performed with Asf1A, Asf1B, the 5'-UTR of TLK1 or -tubulin as probes. (E) Effect of Asf1A, Asf1B and TLK1 triple knockdowns on S-phase progression. The percentages of S-phase cells during RNAi induction were compared among the cells with triple knockdowns, the double knockdowns of Asf1A and Asf1B, and the TLK1 single knockdown. The data represent three independent RNAi induction experiments using the same cell lines.