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Open Access Research article

CRK9 contributes to regulation of mitosis and cytokinesis in the procyclic form of Trypanosoma brucei

Stephane Gourguechon and Ching C Wang*

Author Affiliations

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

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BMC Cell Biology 2009, 10:68  doi:10.1186/1471-2121-10-68

Published: 21 September 2009

Additional files

Additional file 1:

RNAi of the newly identified cyclin and CRK homologues. The data shows the effect of RNAi on each individual novel cyclin and CRK. Procyclic form (29-13) cells were transfected with pZJM vectors carrying short (300-500 bp) fragments of the newly identified cyclin and CRK genes as indicated. Cell lines were selected using phleomycin and cloned. RNAi was induced by addition of tetracycline (10 μg/ml) and cell growth monitored daily using a hemocytometer. Insets show mRNA levels after RNAi for 2 days estimated by semi-quantitative RT-PCR. α-Tubulin was included as a loading control.

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Additional file 2:

Morphology of CRK9 depleted cells. The data shows additional staining and morphology of CRK9 depleted cells to highlight the microtubules and the flagellum. Control and cells depleted of CRK9 by RNAi were fixed, stained with anti-tyrosylated tubulin (marker for basal bodies) YL1/2 and anti-paraflagellar rod antibody (L8C4) and examined by fluorescence microscopy.

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Additional file 3:

Sequence alignment among the newly identified CRK homologues. The figure shows an alignment of the newly identified CRK homologues with the three CRKs 1, 2 and 3 whose functions have been demonstrated in previous studies, using the MacVector software. Residues important for catalysis, binding or the putative active site are indicated. A cartoon depicting the various domain structures of CRK9 compared with the well characterized CRK1-3 is shown in the top panel.

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Additional file 4:

Predicted structure of CRK9. The figure shows the predicted structure of CRK9, obtained by submitting the sequence of CRK9 to the EasyPred 3D structure prediction server, http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred webcite. The predicted structure of CRK1 is shown alongside that of CRK9 (top panel). Some of the important residues are highlighted in colors (red, catalytic aspartic acid, blue, catalytic lysine, dark green, GxGxxG motif required for ATP binding, brown, PSTAIRE motif required for cyclin binding). The structures of the two proteins were also overlaid (bottom panel) and crucial residues highlighted.

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Additional file 5:

CRK9 RNAi in bloodstream form cells. The data presented here shows that CRK9 depletion has no effect on bloodstream form cells. The pZJM-CRK9 vector was transfected into 90-13 bloodstream form T. brucei cells, selected with phleomycin and followed by single cell cloning. RNAi was induced as previously described and cell number monitored daily. Real time quantitative RT-PCR was used to monitor the depletion of CRK9 after 2 days of RNAi (insets); with α-tubulin serving as the loading control.

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Additional file 6:

Localization of CRK9 in bloodstream form cells. The data shows that CRK9 localizes to the nuclei of bloodstream form cells. The pc-CRK9-3HA-BLA plasmid was linearized, transfected into 90-13 bloodstream form T. brucei cells, and stable cell lines were selected using blasticidin (10 μg/ml) and cloned to express CRK9-3HA at the endogenous level. Transfected and control cells were analyzed by Western blotting for the HA tag as indicated (A). The blot were stripped and probed with anti-α-tubulin antibody to verify equal sample loading. Localization of CRK9 was determined by fixing and staining the transfected cells with a FITC-conjugated anti-HA antibody (B). DAPI was used to visualize the nucleus and kinetoplast and merged images were created using the imageJ software.

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