Constructs and Protein Purification
The HSET cDNA (Genbank ID: 68299768
) was a generous gift from Duane Compton (Dartmouth Medical School, Hanover, NH). The cDNA encoding the nonmotor domain of HSET (amino acids 2–304) was subcloned into pGEX1* and p6HGFP-B vectors with the restriction enzymes BamHI and EcoRI to generate glutathione S
-transferase (GST)- and 6-His-green fluorescent protein (GFP)–tagged fusion proteins, respectively. The HSET mutants HSET-NLSa (36KRR to AAA), HSET-NLSb (50KKR to AAA), and HSET N593K were made using the Quikchange site-directed mutagenesis system (Stratagene, La Jolla, CA). pEGFP-HSET and pEGFP-HSET N593K clones were generated by subcloning their cDNA into the pEGFPC1 vector by EcoRI/SalI. The XCTK2 constructs and mutants were described previously (Ems-McClung et al., 2004
). All resulting clones were verified by sequencing.
GST-HSET(2–304) was expressed in Escherichia coli
BL21(DE3) cells and affinity-purified on glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ) as previously described (Ems-McClung et al., 2004
), followed by the gel filtration on a Superose 12 column (GE Healthcare Life Sciences, Piscataway, NJ) equilibrated in column buffer (50 mM MOPS, pH 7.2, 50 mM NaCl, 0.1 mM EDTA, and 0.1 mM EGTA). Fractions were eluted, and sucrose was added to 10%. Fractions were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until use. His-GFP-HSET(2–304) was expressed, purified, and cross-linked to Affi-Gel 10 (Bio-Rad, Hercules, CA) as previously described (Walczak et al., 1997
). Antibodies to the nonmotor domain of HSET were generated by immunizing a rabbit (Covance Research Products, Denver, PA) with GST-HSET(2–304) protein that was first treated with 0.5% glutaraldehyde for 45 min and then quenched with a trace of sodium borohydride for 5 min at room temperature (RT). HSET specific antibodies were then affinity-purified using a His-GFP-HSET(2–304) affinity column as described previously (Walczak et al., 1997
For protein expression of the GFP-fusion proteins XCTK2, XCTK2 NLSa, XCTK2 NLSb, HSET and HSET-N593K, coding sequences were subcloned into pFastBac1-GFP using SacI and KpnI restriction enzymes, and baculovirus was produced using the Bac-to-Bac baculovirus system (Invitrogen, Carlsbad, CA). The GFP-tagged proteins were then expressed in Sf-9 insect cells and purified by conventional chromatography as previously described (Walczak et al., 1997
). The proteins were stored in FPLC buffer (20 mM PIPES, 1 mM MgCl2
, 1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 50 μM Mg-ATP, 400 mM KCl, and 0.1 μg/ml LPC) with the addition of 10% sucrose, flash-frozen in liquid nitrogen, and stored at −80°C.
Nuclear Import and Spindle Assembly in Xenopus Egg Extracts
CSF extract was made from Xenopus laevis
eggs as previously described (Murray, 1991
). For the nuclear import assay, demembranated sperm nuclei were added at a concentration of 500 sperm/μl to CSF extract that was activated by the addition of 0.4 mM CaCl2
for 2 h until most of the sperm had formed a nuclear envelope and appeared round. At this time, a final concentration of 20 nM GFP-XCTK2 or the NLS mutants GFP-XCTK2 NLSa or GFP-XCTK2 NLSb were added to the extract with the preformed nuclei. Ten minutes after the addition of the recombinant proteins, samples (1 μl) were taken and squashed on a microscope slide with 3 μl of spindle fix (60% glycerol, 5 mM HEPES, pH 7.8, 0.1 mM EDTA, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2
, 2 mM CaCl2
, 10% formaldehyde, and 1 μg/ml Hoechst) and immediately imaged using fluorescence microscopy.
For spindle assembly assays, CSF extracts were supplemented with 200 nM X-Rhodamine-labeled tubulin, 500 sperm nuclei/μl, and the indicated recombinant proteins (GFP, GFP-XCTK2, GFP-XCTK2NLSa, GFP-XCTK2NLSb, GFP-HSET, and GFP-HSET N593K) at either a 2.5- or 5-fold molar excess over the endogenous XCTK2 as indicated in the text. The endogenous concentration of XCTK2 in CSF extract is ~20 nM. All fusion proteins were diluted to a 10-fold concentrated stock in CSF-XB buffer, and then equivalent volumes were added to each spindle assembly reaction before spindle assembly for a final 1× concentration. For cycled extracts, CSF extracts were cycled into interphase with the addition of Ca2+
and then back into mitosis with fresh CSF extract as described (Sawin and Mitchison, 1991
; Shamu and Murray, 1992
). For SDS-PAGE, 5 μl of each extract reaction was diluted in 45 μl of sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 4% β-mercaptoethanol, and a trace amount of bromophenol blue) and subjected to Western blot analysis to confirm that equivalent amounts of the proteins were added to the extract. Thirty minutes after initiation of the spindle assembly reaction, 30 μl of each extract sample was diluted in 1 ml of BRB80 (80 mM PIPES, pH 6.8, 1 mM MgCl2
, and 1 mM EGTA) containing 30% glycerol (vol/vol) and fixed by the addition of 1 ml of 30% glycerol, BRB80, and 4% formaldehyde for 20 min at RT. The fixed samples were layered on a 4-ml cushion of BRB80 containing 40% glycerol (vol/vol) and were centrifuged onto coverslips in a Beckman (Fullerton, CA) JS7.5 rotor at 6000 × g
for 15 min. The coverslips were postfixed in −20°C methanol for 5 min and rehydrated in TBS-TX (10 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Triton X-100). DNA was stained by incubation in 1 μg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) diluted in TBS-TX, mounted in anti-fade (90% glycerol, 20 mM Tris-HCl, pH 8.8, and 0.5% p
-phenylenediamine), and sealed with nail polish.
Cell Culture, RNA Interference, Gene Transfection, and Creation of Stable Cell Lines
HeLa cells were cultured at 37°C in Opti-MEM (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). For small interfering RNA (siRNA), HeLa cells at 4 × 104
cells/well were plated into each well of a six-well culture dish, arrested with 2 mM thymidine for 20 h, and then released into fresh media. Two hours after release from the thymidine block, 200 nM Luciferase RNA interference (RNAi) negative control no. 2 oligonucleotide (Dharmacon, Chicago, IL), 200 nM HSET RNAi oligonucleotide to the coding region (UCA GAA GCA GCC CUG UCA A), or 200 nM hNuf2 RNAi oligonucleotide (DeLuca et al., 2002
) was transfected using Oligofectamine (Invitrogen). When comparing knockdown of multiple proteins, an equivalent amount of Luciferase negative control oligonucleotide was cotransfected with the specific oligonucleotide such that the total amount of oligonucleotide transfected was identical for each well. At 24 h after transfection, cells were blocked with 2 mM thymidine for 19 h to synchronize the cells and then released for 11–12 h to allow cells to progress to late G2. The cells were then processed for immunofluorescence, for live imaging, or for Western blot analysis.
To generate the stable cell lines, pEGFP-HSET or pEGFP-HSET N593K were individually introduced into HeLa cells using a calcium phosphate transfection in the presence of 2 mg/ml geneticin (Invitrogen; Rodriguez and Flemington, 1999
). Transfected cells were replated at a density of 1 × 103
cells/plate in a 10-cm plate and grown for 7–10 d with daily changes in media until each colony had ~200 cells. A single colony was isolated with a pipette tip by examining the colony under an inverted fluorescence microscope, transferred to a new dish, and maintained in Opti-MEM containing 1 mg/ml geneticin (Invitrogen). pEGFP-H2B and pmcherry-tubulin plasmids were sequentially introduced into HeLa cells to an established stable expressing cell line as described above. For transient transfections, pEGFP-HSET NLSa or pEGFP- HSET NLSb were introduced into HeLa cells using calcium phosphate transfection and fixed and processed for immunofluorescence 24 h after transfection.
Immunofluorescence and Western Blot Analysis
HeLa cells were fixed in −20°C methanol for 5 min at RT and rehydrated in TBS-TX for 2 min. Cells were blocked in Abdil (TBS-TX, 2% bovine serum albumin, and 0.1% sodium azide) for 1 h at RT or overnight at 4°C. Cells were stained with antibodies against HSET (2.5 μg/μl), Hec1 (1 μg/μl; Novus Biologicals, Littleton, CO), ACA (1:100; Antibodies Incorporated, Davis, CA), or α-tubulin DM1α (1:1000; Sigma-Aldrich) diluted in Abdil for 1 h at RT. Cells were subsequently stained with a 1:50 dilution of donkey anti-rabbit Alexa Fluor 488 (Invitrogen) or donkey anti-mouse TexasRed (Jackson ImmunoResearch Laboratories, West Grove, PA). To visualize DNA, fixed cells were stained with 2 μg/ml Hoechst (Sigma-Aldrich) in TBS-TX.
For Western blot analysis, HeLa cells were collected and lysed in sample buffer at 3000 cells/μl, and Xenopus extract was lysed by 1:10 dilution in sample buffer. 60,000 HeLa cells or 20 μl of extract sample were loaded on a 10% SDS-polyacrylamide gel and transferred to Protran nitrocellulose (Schleicher & Schuell, Waltham, MA). Blots were then probed with DM1α (1:10,000, Sigma-Aldrich), anti-HSET (2.5 μg/ml), anti-GFP (1.9 μg/ml), anti-importin β (1 μg/ml; Sigma-Aldrich), or anti-importin α (1:1000; from Mary Dasso, National Institutes of Health, Bethesda, MD), followed by sheep anti-mouse IgG HRP-linked whole antibody (1:10,000; Amersham, Piscataway, NJ) or donkey anti-rabbit IgG HRP-linked whole antibody (1:10,000; Amersham) and detected with SuperSignal West Pico enhanced chemiluminescence substrates according to the manufacturer's directions.
Immunoprecipitations from Xenopus
egg extracts were performed as described previously (Ems-McClung et al., 2004
). Anti-GFP and nonimmune rabbit IgG were covalently coupled to the Affi-prep protein A beads (Bio-Rad; Harlow and Lane, 1999
). Where indicated, 200 nM purified GFP-XCTK2, GFP-XCTK2-NLSa, or GFP-XCTK2-NLSb was added to the immunoprecipitation reactions. Equivalent volumes of the eluted protein were electrophoresed on 10% SDS-PAGE gels and stained with Coomassie Blue G-250 or transferred to Protran. Western blots were probed as described above.
Immunoprecipitation in HeLa extract was performed as described (Luders et al., 2006
) using anti-HSET antibodies cross-linked to Affi-prep protein A beads. The GFP-HSET overexpressing HeLa cells were plated in a 10-cm plate and treated with 100 ng/ml nocodazole overnight. Cells were trypsinized and diluted with ice-cold Opti-MEM. Cells were centrifuged at 1000 × g
, resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, and 1/1000 LPC) for 5 min on ice using a ratio of 1 ml buffer/10-cm plate. HeLa cell lysates were centrifuged in a TLS 55 rotor at 16,000 rpm for 10 min in a Beckman Optima L-90K Ultracentrifuge. For immunoprecipitations, 400 μl of supernatant were incubated with 50 μl of Affi-prep IgG beads or anti-GFP beads for 5 h at 4°C following the procedure described above for immunoprecipitations in egg extracts.
Cold Treatment for Analysis of Stable MTs
HeLa cells were transfected with the Luciferase control oligonucleotide or HSET RNAi oligonucleotide, and the GFP-HSET HeLa cell line was transfected with the Luciferase control oligonucleotide. At 24 h after transfection, cells were synchronized with 2 mM thymidine for 19 h and released in fresh medium for 11–12 h. The cells in dishes were put on an ice-water bath for 10 min to allow for MT depolymerization and then were fixed with −20°C methanol for 5 min and processed for immunofluorescence as described above.
Preparation of MT Substrates
Guanylyl-(α,β)-methylene-diphosphonate- (GMPCPP; Jena Scientific, Jena Germany) and paclitaxel-stabilized MTs were polymerized from cycled bovine tubulin as previously described (Desai and Walczak, 2001
). Tubulin was clarified at 45,000 rpm for 5 min at 2°C in a TLA 100 rotor (Beckman Coulter) and then polymerized in the presence of 0.5 mM GMPCPP, BRB80, and 1 mM DTT for 30 min. Paclitaxel was added to 20 μM at 20 min after the start of polymerization. The MTs were pelleted at 37°C for 5 min in a TLA100 rotor and resuspended in BRB49 (49 mM PIPES, pH 6.8, 1 mM MgCl2
, and 1 mM EGTA), 1 mM DTT, and 20 μM paclitaxel.
ELIPA ATPase Assays
ATPase assays were performed using the Enzyme Linked Inorganic Phosphate Assay (ELIPA) kit (Cytoskeleton, Denver, CO) to detect Pi
release. For these experiments, 250 nM GFP-HSET or GFP-HSET N593K were incubated with 0.62 mM MgATP, 50 mM KCl, BRB80, and varying concentrations (0–3 μM) of GMPCPP- and paclitaxel-stabilized MTs (doubly stabilized). The rate of Pi
released was measured using the SpectraMax190 (Molecular Devices, Sunnyvale, CA) with an absorbance of 360 nm at 30-s intervals for 30 min. Data were collected and analyzed from at least three independent experiments and fit to Equation 1
using Prism (GraphPad Software, San Diego, CA) to determine the kcat
of GFP-HSET or GFP-HSET N593K in the presence of MTs. The plots represent the average ± the SE of these experiments.
represents the maximum binding, MTt
represents the total tubulin concentration, and Km
represents the concentration of GFP-HSET to reach half-maximum binding.
HSET MT Cosedimentation Assays
The cosedimentation assays were performed similarly to Hertzer et al. (2006)
using doubly stabilized MTs. Equal molar concentrations (0.35 μM) of purified protein (GFP-HSET or GFP-HSET N593K) were incubated in BRB20 (20 mM PIPES, pH 6.8, 1 mM MgCl2
, and 1 mM EGTA), and 63 mM KCl with increasing concentrations of doubly stabilized MTs (0–0.7 μM). The reactions were pelleted at 45,000 rpm in a Beckman TLA 100 rotor. The supernatant was removed and mixed with an equal volume of 2× sample buffer. The pellet of each reaction was resuspended in a volume of 2× sample buffer equal to the supernatant and mixed with an equal volume of BRB80. Equal volumes of supernatant and pellet samples were analyzed by SDS-PAGE and stained with colloidal Coomassie Blue. The concentration of GFP-HSET partitioning between the supernatant and pellet fractions was quantified by densitometry of the gel using Image J (http://rsb.info.nih.gov/ij/
; NIH, Bethesda, MD). Data collected and analyzed from at least three independent experiments were fit to a two-site quadratic binding equation (Equation 2
; Wang and Jiang, 1996
) using Prism (GraphPad Software) to determine the apparent Kd
of GFP-HSET or GFP-HSET N593K for MTs. The plots represent the average ± the SE of these experiments:
where Mt · E is the concentration of enzyme binding to MTs in nanomolar, E0
is the total amount of enzyme bound in nanomolar, E is the total enzyme concentration, MTt
is the total tubulin concentration, Kd1
is the dissociation constant for site 1, and Kd2
is the dissociation constant for site 2.
For HSET RNAi imaging, GFP-H2B/mcherry-tubulin HeLa cells were plated at 4000 cells/well for both control and HSET RNAi in 96-well BD Falcon imaging plates (Bedford, MA). HSET RNAi was performed using the same method described above except that the volumes of all reagents were normalized based on the smaller surface area of the plates. At 24 h after HSET RNAi, cells were blocked by 2 mM thymidine for 16 h and released in fresh medium for 9 h. Cell plates were placed in BD Pathway 855 and imaged at 20–40-ms exposure time at 5-min intervals ON. For imaging of HSET overexpressing cells, the GFP-HSET overexpressing cell line was plated at 8000 cells/well in a 96-well BD Falcon imaging plate and transiently transfected with p-mcherry-H2B plasmid using Oligofectamine 2000 (Invitrogen). At 8 h after transfection, cells were imaged using the same conditions as for the HSET RNAi cells. Mitotic progression was analyzed manually using the BD Attovision by recording spindle morphology from frame to frame. Mitotic progression was measured as the time from nuclear envelope breakdown (NEBD) to the de-condensation of daughter chromosomes.
Imaging and Statistical Analysis
All images were acquired on a Nikon Eclipse 90i (Melville, NY), using either a 20× (NA 0.5), 40× (NA 1.0), or 100× Plan Apo objective (NA 1.4) and a CoolSnap HQ CCD camera (Photometrics, Tucson, AZ). The camera and filters were controlled by Metamorph (Molecular Devices, Sunnyvale, CA). Image stacks were collected at 0.5-μm steps through the whole cell volume and then deconvolved using Autodeblur (Autoquant Imaging, Bethesda, MD) for 10–20 iterations. The extract samples were imaged using a 40× plan Apo objective (NA 1.0) and are single-plane images. All images were processed in Adobe Photoshop CS and assembled in Adobe Illustrator CS (San Jose, CA) equivalently for control and experimental samples. The mean spindle length and width were determined from three independent experiments and graphed with the SEM using Excel (Microsoft, Redmond, WA). The histogram of the length data were fit with Gaussian distribution by Prism (GraphPad). The curve was plotted in Excel (Microsoft) and superimposed with the histogram in Adobe Illustrator CS. To measure the fluorescence intensity of GFP-HSET and GFP-HSET N593K, Z series of images were taken with the 100× objective at 0.5-μm step intervals to cover the whole volume of the spindle. After 3D reconstruction in Metamorph, an equivalent 50 × 20-pixel box was drawn both in the spindle close to the pole and in the cytoplasm as the background. Background subtracted fluorescence intensity was correlated with the spindle length and presented in the graph. The interkinetochore distance was determined by measuring the center to center distance of ACA staining of sister kinetochores from 150 kinetochores in 30 cells from three independent experiments. Statistical significance was determined with a Student's t test performed in Excel.