Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Open Biology
Laura WoodWilliam C Earnshaw

Abstract

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1-3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and...Continue Reading

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Citations

Apr 4, 2016·Current Opinion in Structural Biology·Marion E PesentiAndrea Musacchio
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Mar 8, 2017·Proceedings of the National Academy of Sciences of the United States of America·Giulia VargiuWilliam C Earnshaw
Aug 5, 2017·Chemical Reviews·George M Burslem, Craig M Crews

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Methods Mentioned

BETA
gene knockouts
transfection
Fluorescence
flow cytometry
Light
transmission electron microscopy
electron microscopy
light microscopy
co-immunoprecipitation
transfections

Software Mentioned

RStudio
Andromeda
limma
R
S kyline
ioconductor
ell Q uest
SILAC
MaxQuant
ax

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