Tissue culture, siRNA, FACS, and fluorescent protein fusions
HeLa S3 cells were grown in DME supplemented with 10% FCS, 2 mM l
-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37°C with 5% CO2
in a humidified incubator. PP1-γ–EGFP-expressing stable cell line (Trinkle-Mulcahy et al., 2003
) was maintained at 300 µg/ml G418, and GFP-Sds22 cell lines were maintained at 1 µg/ml puromycin. Stable HeLa (Kyoto) EGFP–CENP-A cell line (Jaqaman et al., 2010
) was maintained in 500 ng/ml puromycin.
To increase the yield of mitotic cells, cells were collected by mitotic shake off after 18 h in medium containing 330 nM nocodazole or 10 h after release from double-thymidine block. For thymidine block, cells were grown for 18 h in medium containing 2 mM thymidine, washed twice, and released into fresh medium; after 8 h, 2 mM thymidine was added for another 18 h before being released into fresh medium. Finally, after 10 h, mitotic cells were collected by mitotic shake off.
For experiments with monastrol, cells were incubated for 4 h in 100 µM monastrol (Sigma-Aldrich), washed, and released into equilibrated medium. ZM was purchased from Tocris Bioscience and used at the specified concentrations.
To measure G2/M or sub-G1 fractions, cells were collected, washed in PBS, and fixed/permeabilized in 70% ethanol at −20°C for >30 min before staining with 50 µg/ml propidium iodide in PBS containing 50 µg/ml RNase A and 0.1% Triton X-100. The cell profile was recorded on a flow cytometer (FACSCalibur; BD) using CellQuest Pro software (BD). Data analysis was performed using Flowjo software (Tree Star, Inc.). Clusters of two or more cells were excluded from analysis by gating.
N- and C-terminal fusion proteins of human Sds22 (GenBank accession no. Z50749) with and without a 5× Gly-Ala linker were expressed from a bicistronic vector and selected with 1.5 µg/ml puromycin. Five or more clones of each fusion (C103: Sds22-GFP, D103 GFP-5×GlyAla-Sds22) were selected and analyzed. Transient transfectants (200 ng DNA per 6-wells; Effectene; QIAGEN) were analyzed 48 h after transfection.
Double-stranded Sds22 siRNAi (5′-CUCUCAAAGGAGAUUGUCGUUCAUCC-3′) and medium GC control (Stealth RNAi; Invitrogen) were introduced by oligofectamine (Invitrogen) or alternatively using HiPerFect (QIAGEN) according to the manufacturers’ instructions. Typically, 9 µmol siRNAi was used per 6-well plate; unless stated otherwise, cells were analyzed 48 h after transfection. PP1-specific RNAi duplexes (PP1-α, 5′-CCGCAATTCCGCAAAGCCAA-3′; PP1-β, 5′-TACGAGGATGTCGTCCAGGAA-3′; and PP1-γ, 5′-AACATCGACAGCATTATCCAA-3′) were obtained from QIAGEN. Lipofectamine 2000 was used for cotransfections of mCherry-tubulin and EGFP-Hec1 (provided by D.A. Compton [Dartmouth Medical School, Hanover, NH]) with siRNAs in fluorescence lifetime imaging microscopy (FLIM) FRET experiments.
Sample preparation for proteomic analysis
Cells were lysed in hypotonic buffer (20 mM Tris acetate, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10 mM Na β-glycerophosphate, 5 mM Na pyrophosphate, 1mM Na orthovanadate, 50 mM NaF, 0.1% 2-ME, 0.27 M sucrose, 1% Triton X-100, and 1 µM microcystein), incubated on ice for 15 min, and spun at 1,000 g for 10 min.
GFP fusion proteins were immunoprecipitated with mouse anti-GFP IgG (Roche), prebound, and dimethyl pimelimidate linked to protein G–Sepharose (Bio-Rad Laboratories) or with GFP-Trap (ChromaTek). Beads were preblocked with 0.02% insulin and washed three times with lysis buffer and 0.01% Triton X-100.
Immunoprecipitate eluates were separated on 1D PAGE gel and stained (SimplyBlue; Invitrogen), and protein bands were excised, chopped into ~1 × 1–mm pieces, and destained at RT (2 × 30 min in 10% acetonitrile (ACN)/25 mM triethylammonium bicarbonate buffer [TEAB], pH 8.5). After 10-min dehydration in 100% ACN, proteins in gel were reduced by incubation in 10 mM DTT/TEAB for 60 min at 37°C and alkylated in 50 mM iodoacetic acid/TEAB for 30 min in the dark at RT. For trypsin treatment, gel pellets were dehydrated in 100% ACN and rehydrated on ice (for 1 h) with TEAB buffer containing 10 ng trypsin; excess trypsin was removed, and digestion was performed overnight at 37°C in 50 µl TEAB.
Digested peptides were extracted with 2 × 100 µl 20% ACN/5% formic acid (FA) and once with 100% ACN. Combined extracts were evaporated down to ~20 µl. The final solution was diluted to 100 µl with 3% ACN/0.25% TFA and subjected to cleaning and concentrating procedure on homemade C18 tips. Peptides were eluted from C18 tips in 15 µl 70% ACN/0.1% FA, diluted with 0.1% FA, and concentrated again by evaporation. Liquid chromatography mass spectrometry analysis was performed at Proteomics Facility of the Wellcome Trust Biocentre (University of Dundee, Dundee, Scotland, UK) using a mass spectrometer (Orbitrap; Thermo Fisher Scientific). Combined peak lists were searched against the National Center for Biotechnology Information database using the Mascot program (version 2.2; Matrix Science).
For immunofluorescence, cells grown on coverslips were fixed after washing once in 37°C PBS by incubation in 3.7% formaldehyde/PBS, pH 6.8, two times for 5 min at 37°C. Cells were permeabilized in PBS/0.1% Triton X-100 for 10 min at 37°C, blocked (2% BSA in TBS/0.1% Triton X-100 and 0.1% normal donkey serum) for 40 min at RT, incubated with primary antibodies for 1 h, washed (TBS/0.1% Triton X-100), and incubated with secondary antibodies for 45 min. If required, cells were stained with 0.1 µg/ml DAPI. After a final set of washes, cells were mounted in p-phenylenediamin/glycerol homemade mounting medium (0.5% p-phenylenediamine, 20 mM Tris, pH 8.8, and 90% glycerol). For phospho-specific antibodies, heat-inactivated blocking solution (30 min at 55°C) and wash and fixing solutions were supplemented with 500 nM microcystin and 80 nM okadaic acid. For pHec1 antibody, cells were permeabilized for 2 min in PHEM buffer and 0.2% Triton X-100 and fixed in PHEM and 3.7% PFA.
For aqueous chromosome spreads, cells were grown on coverslips in the presence of 330 nM nocodazole for 18 h, swelled in 60% water and 40% medium for 7 min, and spun down onto coverslips for 4 min at 1,000 g before being processed for immunofluorescence.
Anti-AIM1 monoclonal antibody (BD) was used at 1:200 dilution. Affinity-purified anti–phospho-MCAK (Andrews et al., 2004
) and Sds22 polyclonal antibodies were diluted to 1 µg/ml. Anti-phospho–Ser55-Hec1 antibody was used at a 1:15,000 dilution and anti-phospho–Ser44-Hec1 was used at 1:3,000. Rat anti–α-tubulin (AbD Serotec) and mouse α-Hec1 (Abcam) were used at a 1:500 dilution. Human ACA (provided by S. Marshall and Tayside Tissue Bank, University of Dundee), anti-phospho–CENP-A (BD), sheep anti-BubR1 (provided by S. Taylor, University of Manchester, Manchester, England, UK), and anti–phospho-T232 aurora B (Rockland Scientific) were diluted at 1:1,000. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (FITC, Texas red, TRITC, or Cy5) or Invitrogen (Alexa Fluor 488 and 568). Polyclonal sheep anti-PP1 antibody was provided by P.T. Cohen (University of Dundee).
To generate Sds22-specific antibodies, full-length human Sds22 was expressed as a GST fusion in bacteria, purified on a glutathione column, cleaved from the GST tag, and injected into rabbits. Antisera were purified over recombinant full-length MBP-Sds22 bound to an amylose column.
Fixed imaging was performed on a microscope (DeltaVision Core; Applied Precision) built around a stand (IX70; Olympus) with a 100× 1.4 NA lens and a camera (CoolSNAP HQ; Photometrics; Andrews et al., 2004
; Porter et al., 2007
). Fixed cells on No. 1.5 coverslips were mounted in 0.5% p
-phenylenediamine in 90% glycerol. Optical sections were recorded every 0.2 µm. 3D datasets were deconvolved using constrained iterative restoration (Swedlow et al., 1997
; Wallace et al., 2001
) as implemented in SoftWoRx software (Applied Precision). Hec1–Hec1 distances for each condition were measured in SoftWoRx for clearly distinguishable metaphase centromere pairs lying along the pole to pole axis using individual optical sections from 3D datasets.
Image intensity analysis for fixed image data was performed in OMERO software (Swedlow et al., 2009
). A region covering all of the kinetochores at mitotic plate was defined, and intensities through all sections were summed and divided by the area of the region of interest for each channel. An intensity value per area for the background obtained in a similar fashion was subtracted, and relative intensities were expressed as a ratio relative to a general kinetochore marker (ACA or Hec1).
For analysis of cell cycle progression, HeLa cells expressing CENP-A–GFP were imaged in 35-mm glass-bottom dishes (Microwell; MatTek) or cover glass (Labtek) starting 48 h after transfection. Datasets (512 × 512 pixels with 2 × 2 binning, 0.05-s exposure, and five z sections spaced by 0.5 µm) were acquired every 5 min on a microscope (DeltaVision Core) fitted with a 37°C environmental chamber (Solent Scientific) with a 40× 1.3 NA objective and a camera (CoolSNAP HQ). Datasets were deconvolved, and time courses were presented as maximum intensity projections of deconvolved 3D datasets.
Live cell imaging of HeLa cells expressing CENP-A–GFP for measurement of sister pair distances was performed exactly as previously described (Jaqaman et al., 2010
FRET measurements by FLIM were performed on a confocal laser-scanning microscope (Radiance 2100MP; Bio-Rad Laboratories) on a stand (TE2000; Nikon) using a 60×/1.4 NA Plan Apo oil immersion lens (Nikon) equipped with a titanium sapphire laser (Chameleon 1; Coherent) providing femtosecond pulses at a 90-MHz repetition rate. Light shielding and environmental control were achieved using a matt black environmental chamber that surrounded the microscope stage and stand (Solent Scientific) maintaining cells at 37°C and limiting stray light from entering the detectors. Presence of both GFP and mCherry was confirmed using the confocal light path with the 488-nm argon ion and 543-nm HeNe laser lines. Two photon excitation for FLIM was performed at 880 nm with a 600 fps scan speed at 512 × 512 resolution for 60 s, and fluorescent light was collected on a nondescanned detector (5783P; Hamamatsu Photonics) using a 670-nm long-pass dichroic mirror and a 528/50-nm band-pass emission filter, such that only GFP was excited and collected. Time correlated single photon counting was performed using a photon-counting card (SPC830; Becker & Hickl), and subsequent analysis was performed with SPCImage (Becker & Hickl).
3DSIM was performed on a microscope system (OMX version 2; Applied Precision) equipped with 405, 488, and 593 solid-state lasers. Images were acquired using a UPlanS Apochromat 100× 1.4 NA oil immersion objective lens and back-illuminated 512 × 512 electron microscopy charge-coupled device cameras (Cascade II; Photometrics). Samples were illuminated by a coherent scrambled laser light source that had passed through a diffraction grating, thus generating interference of light orders in the image plane to create a 3D sinusoidal pattern with lateral stripes ~0.2 µm apart. The pattern was shifted laterally through five phases and three angular rotations of 60° for each z section. Optical sections were separated by 0.125 µm. Exposure times were typically between 200 and 500 ms, and the power of each laser was adjusted to achieve optimal intensities of between 2,000 and 4,000 counts in a raw image of 16-bit dynamic range at the lowest laser power possible to minimize photobleaching. Each frame acquisition was separated by a 300-ms pause. Multichannel imaging was achieved through sequential acquisition of wavelengths by separate cameras.
Raw 3DSIM images were processed and reconstructed (Gustafsson, 2000
; Schermelleh et al., 2008
). The channels were aligned in the image plane and around the optical axis using predetermined shifts as measured using a target lens and the SoftwoRx alignment tool.
Online supplemental material
Fig. S1 shows biochemical characterization of the interaction between Sds22-GFP and PP1 isoforms and the localization of Sds22-GFP on kinetochores detected with anti-Sds22. Fig. S2 shows the dose-dependent inhibition of aurora B phosphorylation at T232 by ZM. Fig. S3 shows the high resolution localization of total aurora B in control and Sds22-depleted cells using 3DSIM microscopy on an OMX microscope. Fig. S4 shows loss of CENP-A phosphorylation in fixed cells after depletion of individual PP1 isoforms. Fig. S5 shows the effects of Sds22 depletion on phosphorylation of known aurora B substrates and sister interkinetochore distances in living cells. Videos 1–3 show time-lapse imaging of GFP–CENP-A HeLa cells as shown in and maximum intensity projections from 3D time-lapse images. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200912046/DC1