12/24/2022 0 Comments Cellprofiler identify postive cells![]() However, a longer (12 h) exposure to nocodazole will result in lower detectable tubulin fluorescence (Supplemental Figure S1C). A short (15 min) treatment that depolymerizes all the MTs in the cell will not lead to a significant overall decrease in tubulin fluorescence as detected by anti-tubulin antibodies compared with untreated cells with an intact MT array. This effect can be mimicked by exposing cells to short versus long time-course exposure to a MT-depolymerizing drug such as nocodazole. This is because the release of tubulin dimers from MT polymer in cells will suppress tubulin expression, leading to a decrease in overall tubulin fluorescence in cells over the course of several hours. MCAK/Kif2C’s depolymerizing activity has been extensively studied in vitro ( Helenius et al., 2006 Cooper et al., 2010 Friel and Howard, 2011 Gardner et al., 2011), but it can also be quantified in cells by measuring tubulin levels using anti-tubulin antibodies ( Ovechkina et al., 2002 Ogawa et al., 2004 Manning et al., 2007 Montenegro Gouveia et al., 2010 Domnitz et al., 2012 Parker et al., 2018). Mechanistically, this is accomplished by controlling MT assembly rates and lengths in all cellular MTs ( Domnitz et al., 2012 Ertych et al., 2014 Wordeman et al., 2016) and also directly by end modulation and turnover of MTs at kinetochores ( Wordeman et al., 2007 Bakhoum et al., 2009). MCAK/Kif2C uses its depolymerizing activity to suppresses erroneous MT attachments to chromosomes during cell division. This kinesin shares high amino acid identity and conservation within the ATP-hydrolyzing motor domain with minus end– and plus end–directed transport kinesins (Supplemental Figure S1, A and B). MCAK/Kif2C is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. Thus, endogenous MCAK/Kif2C activity in normal cells is tuned to a mean level to achieve maximal suppression of chromosome instability. We also found that increasing WT MCAK/Kif2C protein levels over that of endogenous MCAK/Kif2C similarly increased chromosome instability. Using green fluorescent protein–FKBP-MCAK CRISPR cells we found that one deleterious hot-spot mutation increased chromosome instability in a wild-type (WT) background, suggesting that such mutants have the potential to promote tumor karyotype evolution. We found that a large proportion of these mutations adversely impact the motor. ![]() ![]() This allowed us to rapidly interrogate a number of MCAK/Kif2C motor domain mutations documented in the cancer database cBioPortal. ![]() We improved our workflow using CellProfiler to significantly speed up the imaging and analysis of transfected cells. We found that, despite their distinctly different activities, many mutations that impact transport kinesins also impair MCAK/Kif2C’s depolymerizing activity. This method allows us to score the impact of point mutations within the motor domain. The microtubule (MT)-depolymerizing activity of MCAK/Kif2C can be quantified by expressing the motor in cultured cells and measuring tubulin fluorescence levels after enough hours have passed to allow tubulin autoregulation to proceed. ![]()
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