S1 Supplemental Data Phosphorylation Regulates the Dynamic Interaction of RCC1 with Chromosomes during Mitosis James R. A. Hutchins, William J. Moore, Fiona E. Hood, Jamie S. J. Wilson, Paul D. Andrews, Jason R. Swedlow, and Paul R. Clarke Supplemental Experimental Procedures ture medium (041-95180 M, Invitrogen) plus 10% FCS and 1 peni- cillin/streptomycin immediately prior to analysis. Coverslips were assembled into an FCS2 closed-system, live-cell micro-observation Materials General laboratory reagents were purchased from Sigma or BDH. chamber (Bioptechs) containing CO 2 independent medium. The FCS2 chamber was connected to a thermal control unit so that the Staurosporine and roscovitine were purchased from Calbiochem, dissolved at 10 mM in DMSO, and stored at -20°C. chamber temperature remained at 37°C. The chamber was then mounted onto the stage of a DeltaVision Spectris microscopy work- GFP-RCC1, GFP-27-RCC1, and GFP-RCC1 D182A proteins with an N-terminal hexahistidine (H 6 ) tag were purified as described [S1]. station, which consisted of an Olympus IX70 inverted fluorescence microscope equipped with a computer-controlled stage, a Photo- Phosphorylation site mutants were generated with the QuikChange site-directed mutagenesis kit (Stratagene). For creating RCC1 with metrics CoolSNAP HQ digital camera, and a 10 mW 488 nm solid- state laser and was controlled by softWoRx software (Applied Preci- a C-terminal H 6 -tag, a cDNA was subcloned as a NdeI-XhoI fragment into a modified version of the pET28a vector (Novagen), so that sion) [S2]. Fluorescence images were acquired with a 60 objective lens and a 50 ms exposure. translation was initiated at codon 1 of the RCC1 open reading frame. Expression of RCC1-H 6 protein in E. coli BLR (DE3) cells and purifica- For fluorescence recovery after photobleaching (FRAP) experi- ments, three images of the cell were captured prior to bleaching, tion on Ni-NTA agarose (Qiagen) were performed as before [S1]. AH 6 -importin 3 plasmid was a gift from U. Kutay (ETH Zu ¨ rich, the laser was focused to a diffraction-limited spot, and spot bleach- ing was performed with a single 20 ms stationary pulse. Sixty post- Switzerland). H 6 -importin 3 protein was expressed in E. coli BLR cells and purified on Ni-NTA agarose. An importin  cDNA was a bleach images were captured over the subsequent 40 s, in four phases of increasing interval length: 6 images with 0.15 ms intervals, gift from D. Go ¨ rlich (University of Heidelberg, Germany). The importin  open reading frame was subcloned into the pGEX-4T-1 vector 6 with 0.17 ms intervals, 6 with 0.42 ms intervals, and 42 with 0.83 ms intervals. Analysis of FRAP data was performed with softWoRx (Amersham Biosciences), and the 45-876 truncation was generated by introduction of a restriction site with QuikChange. GST-importin software. Fluorescence intensity values within the bleach spot were determined for each of the time values and expressed as a fraction  proteins were expressed in E. coli BLR cells and purified on gluta- thione-Sepharose 4B beads (Amersham Biosciences). of the mean of the three prebleach values. Fractional intensity values during the recovery phase were fit to the 2D diffusion algorithm of An RCC1-derived pSer11 phosphopeptide CRIAKRRS*PPDA (where S* is phosphoserine) was synthesized and purified by G. Axelrod [S3], yielding recovery half-time (t 1/2 ) and mobile fraction (MF) values. At least ten cells were analyzed for each condition, and Bloomberg (University of Bristol, UK). The phosphopeptide was cou- pled to BSA and used for raising polyclonal antisera in rabbits (Mora- t 1/2 values are expressed  1 standard deviation. For fluorescence loss in photobleaching (FLIP) experiments, three vian Biotechnology, Brno, Czech Republic). -pS11 antibodies were affinity purified against immobilized phosphopeptide coupled to Sul- prebleach images of each cell were taken, and then 100 cycles of laser bleaching (20 ms) and imaging were performed. Image data foLink beads (Pierce Biotechnology), then negatively selected against immobilized GFP-RCC1 coupled to CNBr-Sepharose (Amer- were exported from softWoRx as MPEG files, from which frames were imported into Improvision Openlab software as separate lay- sham Biosciences). ers. Signal intensities within the defined region of interest (nucleus or chromatin) at different time points were determined and ex- Fluorescence Photobleaching (FRAP, FLIP) Human RCC1 constructs in pEGFP-1 (Clontech) were transiently pressed as percentages relative to the first prebleach image. expressed in human U2OS osteosarcoma cells cultured on cov- erslips in Dulbecco’s Modified Eagle medium (DMEM, Gibco) con- Preparation of Cell Extracts Xenopus chromosomal eluates were prepared as described pre- taining 10% FCS, 2 mM glutamine, and penicillin/streptomycin. Cells were transfected with 10 g of plasmid DNA with Lipofectamine viously [S4]. Human HeLa cell extracts were prepared from asyn- chronous cells collected by trypsinization in cold DMEM or from 2000 transfection reagent (Invitrogen). 1–2 days after transfection, the medium was changed for optically clear CO 2 -independent cul- mitotic cells treated with 100 ng/ml nocodazole (Sigma) for 16–18 hr, Figure S1. RCC1 Localizes to Chromatin throughout the Cell Division Cycle Localization of GFP-RCC1 during stages of cell division in live human U2OS cells. The scale bar represents 10 m.