Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

Wenbin Ji; Yibo Luo; Ejaz Ahmad; Song-Tao Liu

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Table S1 . The pMSCV-mCherry-Mis12-MAD1 constructs with wild-type or a mutant MAD1 (K541A, L543A) were gifts from Dr. Tarun Kapoor (Rockefeller University) (26, 28) . Other MAD1 mutants or truncations were cloned or mutated in an intermediate pENTR2B vector (Invitrogen), cut out as a NotI-EcoRI fragment, and then used to replace the MAD1 gene in the pMSCV-mCherry-Mis12 backbone also cut with NotI and EcoRI. Mutagenesis was conducted using QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent). Different fragments of MAD1 were also cloned into a pENTR-D-TOPO vector using TOPO cloning kit (Invitrogen) and then recombined into pDEST15 (for N-terminal GST tag) or pDEST17 (for N-terminal 6×His tag) vectors through LR reactions as instructed for the Gateway cloning system (Invitrogen). MAD2 expression constructs were prepared similarly as before (36) , but with a TEV cleavage site inserted between the tags and second MAD2 codon during pENTR cloning steps. MPS1 was recombined into pDEST10 or pDEST20 and transformed into DH10BAC to prepare recombinant bacmids for Sf9 insect cell transfection. All constructs were confirmed by DNA sequencing. Cell Culture and Transfection. HeLaM, a subline of HeLa (35), was maintained in DMEM with 10% fetal bovine serum at 37 o C in 5% CO 2 . DNA transfection was carried out using TransIT-LT1 reagent (Mirus) following the manufacturer's instructions or using polyethylenimine (PEI) as described before (64) . Sf9 cells were grown at 27 C in SFX medium (Hyclone) in the presence of antibiotics (streptomycin/penicillin). Cellfectin (Invitrogen) was used to transfect bacmids into Sf9 cells. Live Cell Imaging. For determining mitotic durations, HeLa cells grown on No. 1.5 coverslipbottomed 35mm dishes (MatTek) were transfected with different mCherry-Mis12-MAD1 constructs. Usually 24 hr after transfection, live cell imaging was started on an automated Olympus IX-81 microscope to collect phase contrast and RFP images at 15 min intervals using a 60 × objective lens (NA=1.42) while cells were maintained at 37 in a heating chamber. Single-plane images were acquired for up to 13 hr at multiple positions using a CoolSNAP HQ2 camera with 2×2 binning. Student's t-test was used to evaluate the statistical significance between the differences in mitotic durations after different treatment. Nuclear envelope breakdown marks the beginning and appearance of cleavage furrow the end of a mitosis. Some images were collected on a Leica TCS SP8 confocal microscope with a 63 × objective (NA= 1.40) as z-stacks of 1.0 μm. Cell lysates, immunoblotting and immunoprecipitation. These were performed as described before (28, 64) . A list of primary antibodies used in this study is summarized in Table S2 . Recombinant proteins. GST-tagged or His-tagged MAD1 fragments and His-TEV-tagged MAD2 L13A or His-TEV-tagged MAD2 ∆C10 were expressed in E. coli BL21(DE3)-CodonPlus RIPL (Stratagene), normally at 16 o C. His-MPS1 or GST-MPS1 was expressed in Sf9 cells after infection with recombinant baculoviruses. All expressed proteins were purified using GSH-agarose or Probond nickel beads (Invitrogen). His-tagged TEV(S219P) protease(65) was used to cleave His-tag to make untagged MAD2. Peak fractions of eluted proteins were pooled, buffer-exchanged and concentrated using Pierce protein concentrators with 10k cutoff. The storage buffer is 50mM Hepes pH7.5,