Synchronization of procyclic <i>T. brucei</i> and analysis of TbWee1 protein expression during different stages of the cell cycle.

<p>(A) Cells were synchronized with 0.2 mM hydroxyurea for 12 h, then HU was washed out and the cells were stained with propidium iodide and analyzed for 12 hs by flow cytometry performed every 2 h. (B) The percentages of cells in G1, S and G2/M phases were determined by the ModFitLT software. (C) Protein extracts from S, G2/M and G1 were separated by 12 % SDS-PAGE and electroblotted on nitrocellulose membranes. His-tagged recombinant TbWee1 (10 µg) was used as a positive control. Blots were incubated with anti-TbWee1 polyclonal antibody (1:1000) (lanes 1, 3, 5: 20 µg; lanes 2, 4, 6: 40 µg). An antibody recognizing β-tubulin (1:5000) was used as the load control.</p

Effects of TbWee1 knockdown on the procyclic form of <i>T. brucei</i> cells.

<p>(A) Cells of strain 29-13 harboring the TbWee1-RNAi construct were incubated in culture medium with (+ Tet) or without (-Tet) 2.5 µg/ml tetracycline at 28°C. The cell growth rate was monitored daily, and the cell number was plotted in a logarithmic scale. The insets show the intracellular mRNA level after 3 days of RNAi as monitored by Northern blot. RNAr was used as loading control. Western blot of extracts of induced and non-induced cells were analyzed with anti-TbWee1 antibody (Right inset). (B) Time course of RNAi-induced <i>T. brucei</i> procyclic-form. Cells were stained with propidium iodide and subjected to FACS analysis to measure DNA content. The percentages of cells in G1, S and G2/M phases were determined with the ModFitLT software and plotted on the right panel. </p

Amplification and purification of TbWee1-6xHis fusion protein to produce anti-TbWee1 polyclonal antibodies.

<p>(A) SDS–PAGE analysis of lysates obtained from <i>Escherichia coli</i> IPTG.induced (I) and non-induced (NI) cell cultures transformed with pDEST17-TbWee1. Upper panel: Coomassie Brilliant Blue staining. Lower panel: Western-blot analysis with anti-histidine tag antibody (1:5000). The arrow indicates the position of the ~70 kDa recombinant protein. (B) Purification steps of rTbwee1-6xHis fusion protein were monitored by 12 % SDS-PAGE analysis. The lysate was loaded onto a Ni-agarose column. Flow-through (F), washes (W, 1–3), and eluted fractions (250 mM imidazol, E 1–3) were analyzed by 12% SDS-PAGE stained with Coomassie Brilliant Blue. (C) Western blot analysis of rTbWee1 expression with polyclonal anti-TbWee1 antibody. Protein extracts of the <i>T. brucei</i> bloodstream form (lane 1), the <i>T. brucei</i> procyclic form (lane 2) and the recombinant TbWee1-6xHis (lane 3) were separated by 10 % SDS-PAGE and electroblotted on nitrocellulose membrane. Blots were incubated with anti-TbWee1 polyclonal antibody (1:1000) and revealed by chemiluminescence.</p

Rescue of a <i>Schyzosaccharomyces pombe</i> Wee1 mutant by TbWee1.

<p>(A) <i>S. pombe</i> ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which <i>S. pombe</i> Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: <i>S. pombe</i> cells expressing wild type Wee1. (B) DAPI staining of <i>S. pombe</i> cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in <i>S. pombe</i> yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control <i>S. pombe</i> yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.</p

Sequence comparison between TbWee1 and homolog proteins in other species.

<p>(A) Multiple sequence alignment of the catalytic domains of the putative <i>Trypanosoma brucei</i> Wee1 protein with other Wee1-like kinases. Amino acid sequences were aligned using the ClustalW2 program (<a href="http://www.ebi.ac.uk/clustalw" target="_blank">http://www.ebi.ac.uk/clustalw</a>). Identities are indicated by asterisks below the sequence. Conserved substitutions are marked with two vertical dots and semi-conserved substitutions are marked with a single dot. Dashes represent gaps introduced for optimal alignment. The 11 conserved subdomains are designated by Roman numerals [45,46]. The catalytic and activation segments are indicated with a blue and pink box respectively. A black box indicates the conserved EGD motif. Triangles indicate amino acids that are conserved in all known members of the Wee1 kinase family, but not in other eukaryotic protein kinases. Sequences shown are for Wee1A kinase of <i>Trypanosoma </i><i>brucei</i> (TbWee1, JN083854), humans (HsWee1, NP003381.1), mice (MmWee1, NP033542.2), <i>Schizosaccharomyces </i><i>pombe</i> (SpWee1, NP587933.1), <i>Saccharomyces </i><i>cerevisiae</i> (ScSwe1, NP012348.1), <i>Arabidopsis </i><i>thaliana</i> (AtWee1, NP171796.1), <i>Xenopus laevis</i> (XlWee1, NP001081784.1), and <i>Trypanosoma cruzi</i> (TcWee90, JN573306; TcWee570, JN257712). (B) Comparison of TbWee1 with other protein kinases. The position of the putative protein kinase domain is shown in black and numbers represent the percent amino acid identity with this region of the predicted TbWee1.</p

Morphological phenotypes of Wee1-deficient <i>T. brucei</i> procyclic-form cells.

<p>Samples of the TbWee1-depleted cells taken at different times were stained with DAPI and examined by fluorescence microscopy. (A) Analysis of the numbers of nuclei and kinetoplasts as determined by DAPI staining. Data are presented as the mean percentages ±S.E. of the total population counted (> 200 cells in each of three independent experiments). (B) Wee1-deficient cells viewed by phase-contrast and fluorescence microscopy. N: nucleus, K: kinetoplast.</p

Autophosphorylation of recombinant TbWee1-6xHis fusion protein.

<p>Phosphorylation reactions were performed in the absence or presence of 40 µg of rTbWee1-6xHis expressed in the baculovirus system, in a mixture containing 5 µCi [γ-32P]-ATP (6000 Ci/mmol, NEN) for 10 min at 30°C. Reaction products were resolved by 12 % SDS-PAGE and visualized by autoradiography. Membranes were revealed with anti-histidine tag antibody (1:5000) and anti-phosphotyrosine antibodies (1:1000).</p