The Effect of Site-Directed Mutagenesis of Borealin on Its Activity in Cell Division

The effect of site-directed mutagenesis of Borealin on its activity in cell division Student: Tajekesa Blee Supervisor: Dr Sally Wheatley, School of Biomedical Sciences, Queens Medical Centre, University of Nottingham Borealin is a key component of the conserved chromosomal passenger complex (CPC), known for its regulatory roles in mitosis and cytokinesis. Through database mining, we recently discovered that a point mutation in the penultimate residue of borealin which substitutes a histidine residue for a tyrosine (H279Y) is associated with the aggressive brain tumour, glioblastoma multiforme. The aim of this investigation was to determine whether expression of the mutant form affects borealin activity in cell division. Our results showed that while overexpression of wild-type borealin was toxic, the mutated form repressed this toxicity. Thus we conclude that H279Y could contribute to the diseased state, however, whether this is due specifically to its role in mitosis is yet to be verified. Here we hypothesised that a mutation in the wild Introduction: type borealin protein alters its activity. Thus, the Cell division is to be under stringent control for aim of this project was to follow the effect of the proper development and appropriate abnormal, mutated human borealin on its proliferation. If uncontrolled, errors in the cell expression and activity in cultured human cells machinery may lead to excessive cell proliferation during cell division. causing cancer. Borealin, first described in 2004[1] is a core member of the CPC, which is responsible Description of work carried out: for regulatory roles at the centromeres and the DNA cloning and site directed mutagenesis: Wild- central spindle as correct chromosome alignment type human Borealin was cloned into a pcDNA and segregation[1],[2]. Previous database mining vector with a C-terminal GFP tag. BorealinWT-GFP project (International Cancer Genome proteins were expressed in E.coli strain DH5α Consortium, ICGC) in the laboratory discovered overnight at 37°C. The complexes were produced the point mutation on the penultimate residue by copurification of the overnight cell cultures that linked this protein to glioblastoma using QIAprep. PCR was performed using multiforme tumour in human cell lines. This site is Quikchange (Stratagene) on BorealinWT-GFP in intriguing because it is highly exposed, moreover pcDNA for expression in mammalian cells. substituting the Histidine amino acid, at position 279, for a Tyrosine residue (H279Y, Fig. 1), Culture and Transfection of Human Cells: HeLa potentially creates an additional (human cervical cancer) cells were maintained at phosphoregulatory site. 37°C with 5%CO2, in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, penicillin/streptomycin, and fungizome. pcDNA constructs were transfected into HeLa cells using HiPerfect Transfection (QIAGEN). To create cell lines stably expressing the desired constructs, transfected cells were treated with 500μg/ml G418 and GFP-positive colonies were selected 7 days post-transfection. To ensure homogeneity prior to analysis, clones were sorted using fluorescence activated cell sorter (FACS). Fluorescence microscopy: Cells were grown on poly-L-lysine coated coverslips then fixed with 4% formaldehyde and permeabilised with 0.15% Triton. Cells were viewed using a fluorescent microscope (Leica DMRB) fitted with a x40 oil Figure 1: Structure of wild-type Borealin immersion objective. Images were captured using homodimer[3] with C-terminal residues displayed, a cool snap camera and OpenLab software. Chain A: Blue, Chain B: Purple. The novel mutant BorealinH279Y-GFP was produced by mutagenesis of SDS-PAGE and Immunoblotting: the penultimate residue within BorealinWT-GFP Cells were homogenized in RIPA buffer (20 mM construct. Tris–HCl, pH 8.0, 137 mM NaCl, 0.5mM EDTA, 10% glycerol, 1% NP40, 0.1% SDS and 1% sodium deoxycholate) supplemented with a protease

of 4 inhibitor cocktail (CLAP (1/500), AEBSF (1/500)), with live imaging in order to fully investigate nuclease (benzonase, 1/500) and 2mM MgCl2. whether the behaviour of the mutant borealin Cell lysates were centrifuged at 2000rpm for 1 within cells is primarily due to the min to remove insoluble material. Proteins (20 μg phosphorylatable element of the tyrosine in each lane) were separated on 10% SDS– residue. Future experiments may therefore polyacrylamide. involve mutation of a non-phosphorylatable residue to determine attainment of the same Immunoblot analysis was then performed by results in order to confirm that the change in electrotransfer onto Nitrocellulose Membranes activity is not due to e.g. a signalling effect. which were initially blocked overnight with PBS- Tween buffer containing 5% milk powder and Value of the studentship: subsquently probed for 2 hours with two rabbit This opportunity has provided me with great anti-Borealin, Bor92 & Bor93 (generated in experience allowing me to develop confidence in house, 1/500 in 2.5% blocking solution) to reveal a wide range technical skills that will form a solid exogenous BorealinWT-GFP and endogenous foundation for my future career within the Borealin and sequentially with anti-tubulin (B512, Biomedical Sciences. As the project was tailored 1/2000 in 2.5% blocking solution) as a loading to be of relevance to the field that I was initially control. Secondary antibodies were rabbit interested, I have also been able to apply my (Dakocytomation, 1/2000 in 2.5% blocking theoretical knowledge, gained from coursework, solution) and mouse (Dakocytomation, 1/2000 in and have learnt how to integrate knowledge and 2.5% blocking solution) and bands were detected discovery. Although this may have seemed a tiny using ECL (Pierce) and X-ray film (Kodak). drop in the ocean in the developments in scientific research, it has been an invaluable Results and Discussion: experience in my career, and has convinced me A novel mutant BorealinH279Y-GFP construct was to continue working as a scientist. successfully created in the pcDNA vector using site-directed mutagenesis and sequence verified References: (Fig.2). This construct, which also had a C- terminal GFP tag, was used to transfect cultured 1. Gassman, R., Carvalho, A., Henzing, A.J., human cells (HeLa). Localisation data as viewed Richaud, S., Hudson D.F., Honda, R., Nigg, under the microscope showed no change in E.A., Gerloff, D.L., and Earnshaw, W.C. interphase. Localisation of BorealinH279Y-GFP was (2004) Borealin: A novel chromosomal therefore, still in nucleoi as seen in BorealinWT-GFP passenger required for stability of the (Fig.3A-C). As transfection efficiency was quite bipolar mitotic spindle. J. Cell Biol. 166, low, we enriched for BorealinWT-GFP expressing by 179-191 FACS. However, after only two passages post- FACS immunoblotting analysis did not produce strong positive results for the presence of the 2. Zhou, L., Li, J., George, R., Ruchaud, S., mutant proteins within the FACS-sorted Zhou, H., Ladbury, J.E., Earnshaw, W.C., mammalian cells. Consistent with this, transient and Yuan, X. (2009) Effects of full-length transfection of the constructs; BorealinWT-GFP, Borealin in the composition and protein- BorealinH279Y-GFP and BorealinC-termΔ10 (this control protein interaction activity of a binary mutant has a C-terminal truncation which chromosomal passenger complex. removed the last ten residues) (Fig.4A-C) showed Biochemistry 48, 1156-1161 low expression of the wild-type borealin in cells over a 72hr period in comparison to the mutants 3. Bourhis, E., Lingel, A., Phung, Q., which have a higher number of expressing cells. Fairbrother, W.J., Cochran, A.G. (2009) Thus, these initial findings support our hypothesis Phosphorylation of a borealin that mutation in the wild-type borealin changes dimerization domain is required for its expression and therefore, activity within proper chromosome segregation. human cultured cells. Biochemistry 48, 6783-6793

In this case, we can deduce that cells tolerate the 4. BLASTn - Basic Local Alignment Search mutant borealin better than the wild-type Tool (2011) [WWW]< protein, so the mutation may be deleterious in http://blast.ncbi.nlm.nih.gov/Blast.cgi>[Ac patients by having a proliferative effect in the cessed September 2011] tumour. Future directions: From the research, it was established that it is important to carry out transient transfections

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Figure 2: Blast Nucleotide Sequence alignment[4] showing nucleotide substitution by DNA mutagenesis from C in BorealinWT-GFP construct to T in BorealinH279Y-GFP. This mutation caused an amino acid change in the protein that substituted the Histidine to a Tyrosine as required for investigation. (BLASTn, 2011).

Figure 3: BorealinWT-GFP overexpression is localised in the nucleoli. HeLa cells A B C stably expressing the construct were fixed and stained with DAPI (C) to reveal its localisation in the nucleoli (arrows, A). Similarly to BorealinWT-GFP, BorealinH279Y-GFP and BorealinC-termΔ10 also localised in the nucleoli (not shown).

A B Figure 4: BorealinWT-GFPactivity is altered by substitution of Histidine residue for Tyrosine in the penultimate residue. Expression of mutant C borealin conferred a survival advantage over cells expressing the wild- type borealin. Data shown are the results of three different transient transfections 24hrs (A), 48hrs (B) and 72hrs (C) for the three different cell lines expressing the novel constructs BorealinWT-GFP, BorealinH279Y-GFP and BorealinC-termΔ10. They show that, over the investigated time period, cells reduced their tolerance for BorealinWT-GFP so less expressing cells were

of 4 seen. In contrast, cells were tolerant of both mutant constructs with increasing quad events being seen as a reflection of continuing cell division.