Dynamic proteomics in human cancer cells
We developed dynamic proteomics, a system for monitoring the position and amounts of endogenous proteins in individual living human cells. For this purpose we built the Library of Annotated Reporter Cell-clones (LARC). Each cell-line clone contains an endogenous protein fused to yellow fluorescent protein (YFP), expressed from its endogenous chromosomal location with its natural regulation. Labeling with YFP was done by exon-tagging (also known as CD-tagging), where YFP, flanked by splice signals, was delivered into the genome using a retrovirus. YFP is then spliced into the protein as a new exon. Protein identity was established by sequencing the cDNA. The cell line used is the H1299 non-small lung cell carcinoma line. This is a robust cell-line with a nicely spread cell morphology that is highly photogenic for microscopy purposes. Several lines of evidence indicate that over 2/3 of the tagged proteins retain their proper functionality and localization.
The amount and location of each tagged protein is monitored by means of automated time-lapse fluorescence microscopy in controlled CO2 and humidity conditions. Movies of the cells are automatically processed using dedicated image analysis software.
We used dynamic proteomics to study basic biological questions in human and cancer cell biology
- Why is one cell killed by an anti-cancer drug while its neighbor cell survives? Survival correlates with dynamic cell-cell differences in specific proteins. [Cohen 2008]
- Protein localization changes – not protein levels - clearly tell story of cell response to drug. [Cohen 2008]
- Protein noise has memory time of about a cell generation. [Sigal 2006]
- Protein dynamics in drug combinations: linear superposition of single drug dynamics. [Geva 2010]
- Bleach-chase method to measure protein lifetime: About 50% proteins dominated by dilution by cell growth. [Eden 2011]