Functional characterization of MCAK/Kif2C cancer mutations using high-throughput microscopic analysis
| Autor: | Linda Wordeman, Sarah Domnitz, Mike Wagenbach, Yulia Ovechkina, Juan Jesus Vicente |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Mutant Kinesins medicine.disease_cause 03 medical and health sciences 0302 clinical medicine Protein Domains Microtubule Chromosome instability Chromosomal Instability Neoplasms medicine Humans Point Mutation Amino Acid Sequence Molecular Biology Cytoskeleton 030304 developmental biology 0303 health sciences Mutation Microscopy biology Point mutation Cell Biology Transfection Articles HCT116 Cells Cell biology Tubulin 030220 oncology & carcinogenesis biology.protein Kinesin |
| Zdroj: | Molecular Biology of the Cell |
| ISSN: | 1939-4586 1059-1524 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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