| Antigen/Gene or Protein Targets | Mitotrap with FRB domain |
| Parental Line | HeLa |
| Host | Human |
| Tissue | Cervix |
| Disease Keywords | Protein transport; cell trafflicking machinery; hereditary spastic paraplegias |
| Relevance |
The HeLa Mitotrap cell line can be used to determine the function of a protein by by rapamycin induced inactivation through rerouting of the protein to the mitochondria.This type of approach, a “knocksideways,” should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days. FRB refers to a protein containing FKBP12 and Rapamycin Binding (FRB) domains within the mTOR (mammalian Target of Rapamycin) protein. The Hela-Mitotrap cell line can be transfected to express the target protein of interest genetically modified to have an FKBP domain. Upon treatment of the cells with rapamycin target proteins containing an FKBP domain will dimerize with FRB domains. The resulting protein complexes are sequestered to the mitochondria. The knocksideways system inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically 3 to 4 orders of magnitude faster than a knockdown. Because a knocksideways is so rapid (typically 10 min), one can investigate the immediate consequences of inactivating a protein of interest. This often leads to more clear-cut results than those obtained with a knockdown or knockout, because there is much less time for indirect and/or compensatory events to occur. |
| Production Details | HeLa-Mitotrap expresses a mitochondrial trapping construct with an FRB domain and is maintained in hygromycin-containing medium. The other three cell lines also express Mitotrap, and in addition they express a protein of interest with an FKBP domain attached, which can be trapped onto mitochondrial when rapamycin is added. The three proteins of interest are Ap1g1 or gamma-adaptin; Ap2a2 or alpha-adaptin isoform C; and GGA2. |
| Conditional | Yes |
| Conditional Description | Rapamicin-induced knocksideways |
| Research Area | Cellular Transport, Genetic Studies Tools |
| Recommended Growing Conditions | HeLa cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. Cells were transfected using GeneJuice. Rapamycin was used at 200 nM, vehicle was ethanol (0.1%). MLN8237 was used at 1 µM, vehicle was DMSO (0.01%). |
| Cellosaurus ID | CVCL_AR66 |
Invented by Margaret Robinson at University of Cambridge
Invented by Margaret Robinson at University of Cambridge
Invented by Margaret Robinson at University of Cambridge
Cheeseman et al. 2013. J Cell Sci. 126(Pt 9):2102-13. PMID: 23532825.
Hirst et al. 2012. Curr Biol. 22(18):1711-6. PMID: 22902756.
Robinson et al. 2010. Dev Cell. 18(2):324-31. PMID: 20159602.
Robinson et al. 2013. Curr Protoc Cell Biol. 61:15.20.1-7. PMID: 24510805.
Cheeseman et al. 2013. J Cell Sci. 126(Pt 9):2102-13. PMID: 23532825.
Hirst et al. 2012. Curr Biol. 22(18):1711-6. PMID: 22902756.
Robinson et al. 2010. Dev Cell. 18(2):324-31. PMID: 20159602.
Robinson et al. 2013. Curr Protoc Cell Biol. 61:15.20.1-7. PMID: 24510805.
|
Margaret Robinson |
|
|
Jennifer Hirst |
