Stout JR et al. (2006), Deciphering protein function during mitosis in ...

XB-ART-34505

BMC Cell Biol 2006 Jan 19;7:26. doi: 10.1186/1471-2121-7-26.

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Deciphering protein function during mitosis in PtK cells using RNAi.

Stout JR , Rizk RS , Kline SL , Walczak CE .

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Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed. By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency approximately 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. P-MCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest. This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells.

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Species referenced: Xenopus laevis
Genes referenced: aurkb kif11 kif2c ptk2 ptk2b

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	Figure 1. P-MCAK is highly similar to H-MCAK. An alignment of P-MCAK, H-MCAK and X-MCAK proteins is shown. All 3 proteins share significant identity in the N-terminal centromere targeting domain (blue brackets) and the centrally located catalytic core (red brackets). The major Aurora B phosphorylation sites that regulate centromere-targeting and microtubule depolymerization activity are also conserved and are indicated in green.
	Figure 2. Fixed analysis of P-MCAK knockdown by RNAi shows defects in spindle assembly and a mitotic delay in prometaphase. PtK2 cells were transfected with a 21 bp siRNA specific to P-MCAK and then processed after 72 hrs for immunofluorescence and for immunoblotting. (A) Immunoblot of serial dilutions of either control or MCAK RNAi cell extracts. K indicates the number of cells in thousands loaded in that lane. (B, C, D, E) Immunofluorescence images of control or MCAK RNAi cells show loss of MCAK staining at centromeres. (F-G) Immunofluorescence images of control or MCAK RNAi cells that show an increase in microtubule (green) density during prometaphase as well as an increase in chromosomes (blue) lingering near the poles (arrows) when MCAK levels are knocked down. (H, I) Quantification of mitotic defects caused by MCAK RNAi. At 72 hrs post-transfection, cells were quantified to determine the specificity of defects seen. (H) Knockdown of MCAK causes a prometaphase delay in mitosis. (I) Knockdown of MCAK causes an increase in cells with increased microtubule density. All data represent mean +/- SEM for 6 experiments. N = 600 cells for mitotic index and N = 100 cells for mitotic phenotype counts for each experiment. Scale bar, 10 μm.
	Figure 3. The congression and segregation defects scored in MCAK inhibited cells are displayed. Live imaging of RNAi knockdown and antibody inhibition of P-MCAK show an increase in congression defects, and an increase in segregation defects compared to control RNAi knockdown and IgG injected cells. (A, B) Series of panels from time-lapse phase contrast microscopy of different movies of either control or MCAK inhibited cells that illustrate the different mitotic defects that were scored in all cells. Images chosen are to represent a particular morphological defect and include images taken from both antibody injection experiments and RNAi experiments. (A) Alignment defects and (B) segregation defects. In each panel, the arrow points to the chromosome of interest, and the line indicates the trajectory of chromosome movement and can serve as a reference point for the extent of chromosome movement. Scale bar, 10 μm.
	Figure 4. P-Eg5 and H-Eg5 share significant identity in their catalytic domains. A series of degenerate oligonucleotides were used to clone a segment of both the H-Eg5 and P-Eg5 cDNA by RT-PCR. (A) An alignment of P-Eg5 and H-Eg5 DNA is shown. The sequence chosen for the 21 bp siRNA is outlined in green.
	Figure 5. Knockdown of Eg5 causes an increase in monopolar spindles. Either PtK2 (A, B, C, D) or HeLa (E, F) cells were transfected with the identical 21 bp siRNA to knockdown Eg5 levels. (A, B, E) Control cells stained to visualize microtubules (green), Eg5 (red) and DNA (blue). Eg5 knockdown cells (C, F) or cells that were treated with the Eg5 inhibitor monastrol (D) were stained to visualize microtubules (green), Eg5 (red) and DNA (blue). Knockdown of Eg5 levels resulted in cells with monopolar spindles and a mitotic delay as reported previously. (G) Quantification of the percentage of RNAi cells with bipolar versus monopolar spindles in either buffer, negative control siRNA, Eg5 siRNA, or monastrol treated cells for PtK2, HeLa and PtK1 cell lines. (H) Quantification of the mitotic index in PtK2, HeLa, and PtK1 cells transfected with buffer, control siRNA, or Eg5 siRNA, or treated with monastrol. All data represent mean +/- SEM for 3+ experiments. N = 600 cells for mitotic index and N = 100 cells for mitotic phenotype counts for each experiment. Scale bar, 10 μm.

References [+] :

Andersen, Mitotic chromatin regulates phosphorylation of Stathmin/Op18. 1997, Pubmed, Xenbase