Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 1

Fig. 1 Generation and characterisation of cell lines expressing mAID-SMASh-tagged cyclin A2 or B1

(A) Schematic of protein degradation using AID and SMASh systems. The SMASh tag consists of the self-cleaving viral HCV NS3 protease fused in cis to a constitutive destabilising peptide. The tag detaches itself from the target protein in the off condition. Upon inhibition by the protease inhibitor, asunaprevir (Asv), the tag remains on the target protein and exposes it to the cellular degradation machinery, while the old protein is depleted depending on its specific half-life. The AID-tag relies on the expression of the plant F-box protein TIR1 that combines with mammalian Skp1 and Cul1 to form a functional SCF E3 ligase that is activated to ubiquitylate the AID degron tag upon stimulation by the hormone Auxin (IAA). In the case of the double tag combination newly synthesised proteins are depleted by the two independent degradation systems. Old proteins are destabilised by the mAID-tag (mini-AID).

(B) Knock-in strategy for inducible OsTIR1 expression at the human Rosa26 locus, and endogenous cyclin A2 and B1 protein tagging. We integrated mAID-SMASh-T2A-neomycin in frame at the C-terminus of the coding sequence of cyclins A2 and B1. The chosen length for the homologous regions for gene targeting was approximately 1kb.

(C) To verify gene targeting primers were designed in the genomic locus and in the cassette (indicated in (B)) and used to amplify genomic DNA from different cell lines: wild-type RPE-1, RPE-1 expressing OsTIR1, CycA2dd, CycB1dd, and primers amplifying a region of the Kif23 gene were used as a positive control (see Material and Methods section for primer sequences).

(D; E) (D) Representative immunoblots and (E) quantitation showing timecourse of induced degradation of double degron tagged cyclin A and cyclin B (A2dd, B1dd). * indicates crossreacting band. (n=3 experiments, s.d. indicated by error bars). Doxycycline/IAA/Asv (DIA, see Methods) was added to asynchronous cells for indicated time before extract preparation and immunoblotting.

(F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA or both (DIA, see methods) then collected for western blot analysis. Doxycycline was added 2 hours before addition of IAA or Asv/IAA. To determine protein degradation, either cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1 antibody.

(G) Timecourse of DIA induced cyclin A and B degradation, cross checking levels of each mitotic cyclin. Treatments were performed as for DIA treated panels in (E). Cell extracts were checked for both cyclin A2 and B1 levels following DIA addition (* indicates non-specific band).

(H) Comparison of DIA and siRNA depletion. Cells were treated with DIA for four hours or pre-transfected with CycB1 SMARTpool siRNAs 48 hours earlier. Cell extracts were analysed by immunoblotting for cyclin B1 (L.E, long exposure, S.E. short exposure) using the same mouse monoclonal antibody as in (E) and (F).

(I) Assaying cyclin B1 depletion in mitotic cells using a rabbit mono-clonal antibody. Extracts from asychronous or mitotic cells were collected and analysed by immunoblotting using the indicated antibodies.