Technical Reference #1904

1904.

Overexpression of the E2 ubiquitin–conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation Janine H. van Ree; Karthik B. Jeganathan; Liviu Malureanu; and Jan M. van Deursen, Mayo Clinic College of Medicine, Journal of Cell Biology, 188(1904), (2010)
Link To Paper

Materials & Methods:
Live cell imaging- Nocodazole challenge assays were performed as follows. MEFs were first transduced with a lentivirus encoding an mRFP-tagged H2B to allow visualization of chromosomes by fluorescence microscopy. Cells were then seeded onto 35-mm glass-bottomed culture dishes (MatTek Corporation) and cultured in DME/10% FBS. Approximately 24 h later experiments were performed using an AxioObserver Z1 system (Carl Zeiss Inc.) with CO2 Module S TempModule S Heating Unit XL S Plan-Apochromat 63× NA 1.4 oil DICIII objective a camera (AxioCam MRm; Carl Zeiss Inc.) and AxioVision 10.6 software (Carl Zeiss Inc.). The imaging medium was DME/10% FBS. The temperature of the imaging medium was kept at 37°C. The exposure times in nocodazole challenge experiments were identical among experiments. Nocodazole was added to a final concentration of 100 ng/ml. Cells undergoing NEBD were marked and monitored at 30-min intervals to determine when they decondensed their chromosomes. The duration of arrest in mitosis which is defined as the interval between NEBD (onset of mitosis) and chromatin decondensation (exit from mitosis without cytokinesis) was then calculated and plotted. The time at which 50% of the cells had exited mitosis was used for comparison (see also Baker et al. 2006). For mitotic timing experiments the time interval between NEBD and anaphase onset was measured as H2B-mRFP–positive cells progressed through an unchallenged mitosis. For chromosome missegregation analysis H2B-mRFP–positive cells progressing through an unchallenged mitosis were followed at interframe intervals of 3 min. Monitoring of securin stability by live cell imaging was performed as described previously (Malureanu et al. 2009) with the exception that pECFP-securin was used instead of pYFP-securin (transgenic MEFs already express EGFP). The pECFP-N1-securin expression vector that was used was produced by ligating the BamHI and XhoI securin cDNA fragment of pYFP-N1-securin (from J. Pines Gurdon Institute Cambridge England UK) into the BamHI and XhoI sites of pECFP-N1. H2B-mRFP– expressing UbcH10+/+ and UbcH10T2/T2 MEFs were nucleofected with 2 μg pECFP-N1-securin expression plasmid. For image processing we used Axio- Vision 10.6 software and PowerPoint (Microsoft) for Mac.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
UbcH10 transgenic MEFs