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Functional characterization of MCAK/Kif2C cancer mutations using high-throughput microscopic analysis
Mike Wagenbach1, Juan Jesus Vicente1, Yulia Ovechkina1
1Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195.
Abstract:
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. This method allows us to score the impact of point mutations within the motor domain. We found that, despite their distinctly different activities, many mutations that impact transport kinesins also impair MCAK/Kif2C's depolymerizing activity. We improved our workflow using CellProfiler to significantly speed up the imaging and analysis of transfected cells. This allowed us to rapidly interrogate a number of MCAK/Kif2C motor domain mutations documented in the cancer database cBioPortal. We found that a large proportion of these mutations adversely impact the motor. 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 also found that increasing WT MCAK/Kif2C protein levels over that of endogenous MCAK/Kif2C similarly increased chromosome instability. Thus, endogenous MCAK/Kif2C activity in normal cells is tuned to a mean level to achieve maximal suppression of chromosome instability.
Insights
Mutations in the microtubule-depolymerizing kinesin MCAK/Kif2C can impair its function and promote chromosome instability. Its activity is finely tuned in cells to prevent errors in chromosome segregation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule dynamics are crucial for cell division.
- MCAK/Kif2C is a key motor protein regulating microtubule depolymerization.
- Dysregulation of MCAK/Kif2C is implicated in cancer development.
Purpose of the Study:
- To quantify the microtubule (MT)-depolymerizing activity of MCAK/Kif2C.
- To assess the impact of motor domain mutations on MCAK/Kif2C function.
- To investigate the role of MCAK/Kif2C in chromosome instability.
Main Methods:
- Expression of MCAK/Kif2C in cultured cells.
- Measurement of tubulin fluorescence levels.
- Utilizing CellProfiler for high-throughput imaging and analysis.
- Analysis of MCAK/Kif2C mutations from the cBioPortal cancer database.
- Employing CRISPR technology for targeted gene editing.
Main Results:
- Many mutations affecting transport kinesins also impair MCAK/Kif2C depolymerizing activity.
- A significant proportion of cancer-associated MCAK/Kif2C mutations adversely affect motor function.
- A specific hot-spot mutation increased chromosome instability in a wild-type background.
- Elevated levels of wild-type MCAK/Kif2C also increased chromosome instability.
Conclusions:
- MCAK/Kif2C's depolymerizing activity is sensitive to specific motor domain mutations, including those found in cancer.
- Certain MCAK/Kif2C mutations can promote chromosome instability, potentially driving tumor evolution.
- Endogenous MCAK/Kif2C levels are precisely regulated to maintain genomic stability, suggesting a critical threshold for its function.
