Images were cropped in Adobe Photoshop 6.0, and then sized and placed in figures using Adobe Illustrator CS2 (Adobe Systems). Live-Cell Imaging. Live cell ...
Current Biology, Volume 19
Supplemental Data Cdk1 Negatively Regulates Midzone Localization of the Mitotic Kinesin Mklp2 and the Chromosomal Passenger Complex Stefan Hümmer and Thomas U. Mayer
Supplemental Experimental Procedures
Cloning, Expression, and Purification of Recombinant Proteins Mklp2, Mklp1 and INCENP were PCR-amplified from a human testis cDNA library (Invitrogen). The amplified cDNA was cloned into pCS2-GFP (Amersham) and pQE80 6xHis (Qiagen) plasmids. T59E and T59V-mutant of INCENP was created using the QuikChange protocol (Stratagene). Mklp2 fragments contain the following amino acids: Mklp2NT (aa 1-508), Mklp2CT (aa 508-890). In Cyclin BK
to R
all lysines were
changed to arginine. Cyclin B delta 90 was expressed and purified as described previously [1].
Antibodies mouse α-Aurora B (AIM1) (BD Biosciences), rabbit α-INCENP, mouse α-INCENP, rabbit α-GFP, mouse α-GFP and rabbit α-histone H3 (Abcam), rabbit α-PRC1 (Santa Cruz), rabbit α-myosin IIB, and mouse α-αtubulin (Sigma-Aldrich), human CREST autoimmune serum (Immunovision). Rabbit antibodies were raised against bacterially expressed His-Mklp1 (aa 1-493) and His-Mklp2 (aa 1-508). Primary antibodies were detected with Alexa-Fluor-488 and Alexa-Fluor-568-conjugated goat α-mouse or goat α-rabbit antibodies or with Alexa-Fluor-647 goat α-human antibody (Molecular probes) and for immunoblotting with horseradishperoxidase-conjugated α-mouse or α-rabbit antibodies (Bio-Rad).
Cell Culture and Synchronization HeLa S3 cells were grown at 37 °C under 5 % CO2 in DMEM (Invitrogen). In order to enrich cells in Anaphase, cells were pre-synchronized at G1/S using 5 mM thymidine (Sigma) in cell culture medium for 24
h and released in the presence of 100 nM Nocodazole (Sigma) for 12 h to obtain mitotic arrested cells. Cells harvested by shack-off were released by washing and re-plated for 60 min to enrich cells in anaphase. For drug treatment, cells were arrested in 30 µM VS-83 for 2h, followed by 1 µM taxol for 30 min and finally 100 µM roscovitine and 20 µM MG132 (Sigma) for 25 min. DMSO was used as solvent control in all cases.
Transient Transfections, RNAi, and RNAi Rescue siRNA
duplexes
targeting
INCENP
(5´-GGCTTGGCCAGGTGTATAT-3´)
and
Mklp2
(5’-
CCACCTATGTAATCTCATG-3’) were obtained from Dharmacon Research. As a control, a duplex (GL2) targeting luciferase was used [2]. For rescue experiments, Plasmids and RNAi duplexes were transfected simultaneously using FUGENE6 (Roche) and Oligofectamine (Invitrogen), respectively. After 8 h medium was replaced and 5 mM thymidine and RNAi duplexes were added. Cells were released after 18 h and then either analyzed by life cell imaging (6 h), fixed and stained for immunofluorescence or processed for Western blotting (10 h).
Immunofluorescence (IF) Microscopy Immunofluorescence microscopy was performed using a Zeiss Axioplan II microscope with a 63x oil immersion objective. For high-resolution images, a Deltavision microscope (Olympus IX71, PlanApo 60X/1.40 oil immersion objective, Cool SNAP HQ camera) was used for collecting 0.2 µm distanced optical sections in the z axis. Images were acquired at about 22°C. Images at single focal planes were processed with a deconvolution algorithm, and optical sections were projected into one picture by using Softworx software (Applied Precision). Images were cropped in Adobe Photoshop 6.0, and then sized and placed in figures using Adobe Illustrator CS2 (Adobe Systems).
Live-Cell Imaging Live cell imaging was performed in CO2-independent media (DMEM) and a heated chamber was used to keep cells at 37°C. A Zeiss Axiovert microscope (Zeiss) with a Plan Apo 20x/0.95 objective was used and
data were collected and processed by MetaMorph software. Images were captured with 50-100 millisecond exposure times in 5 minutes intervals for 18 hours.
Cell Extracts, Co-immunoprecipitations, and Immunoblotting For co-immunoprecipitations, HeLa cells were harvested and lysed as described previously [3]. The antibodies for Mklp2 and GFP were coupled to Affi-Prep Protein A Support beads (Bio-Rad) or Protein G beads (Pierce), incubated with the cleared extract and bound proteins were precipitated. For the cdk1 kinase assay Mklp2-beads were incubated with 5 ng/µl cdk1/Cyclin B (Millipore) and as control the kinase was heat inactivated or inhibited by 1mM roscovitine (Sigma). For MT pelleting experiments, cells lysed and subcellular fractioning was performed as described previously [4]. Cyclin Β Δ90 or dialyses buffer as control was added during lyses. MT pelleting from the cytosolic fraction was performed as described previously [5].
Figure S1. The Phosphomimic Mutant of INCENP (T59E) Complements the Function of the CPC in Early Mitosis (A) Domain structure of INCENP. GFP-tagged constructs used in this study are shown below. The targeting region of the applied siRNA-duplexes is indicated by an arrow. (B) Western blots of INCENP- or Gl2-depleted HeLa cells transfected with the indicated GFP-INCENP constructs. Asterisk and arrow indicate ectopically expressed GFP-INCENP variants and endogenous INCENP, respectively. The α-myosin IIB Western blot served as a loading control. (C) Quantification of live-cell recordings of Gl2- or INCENP-RNAi HeLa cells transfected with the indicated constructs. For each condition at least 4 independent experiments were performed analyzing 120 cells at minimum. The time from nuclear envelope breakdown (NEB) to anaphase onset was analyzed and the data are presented in a box blot, showing the median (line), the percentile 25/75 (Box) and the percentile 10/90 (bars). (D) Quantification of the mitotic index of INCENP-RNAi HeLa cells transfected with indicated constructs and treated with 100 nM taxol for 14 hours after releasing the cells from a thymidine-arrest. For each condition three independent experiments were performed analyzing at least 100 cells per experiment. Data were averaged and error bars represent standard deviations (SD). (E) HeLa cells transfected with the indicated siRNA duplexes and rescue constructs were fixed and survivin is shown in red, GFP in green, and DNA in blue. Bar, 10 µm. (F) Lysates (input) were prepared from HeLa cells transfected with the indicated Mklp2 fragments followed by immunoprecipitation of the fusionproteins with GFP antibodies. Western blots were probed with GFP, Aurora-B, and α-tubulin antibodies. Asterisk marks a protein crossreacting with the GFP antibody. (G) Mklp2 was immunoprecipitated from anaphase extracts prepared as described and the precipitate was incubated with ATP and recombinant Cdk1/cyclin B for 30 minutes at 25°C in the presence or absence of roscovitine. Bead associated Aurora-B was detected by Western blotting.
Supplemental References 1.
Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P., and Kirschner, M.W. (2001). Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715-726.
2.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
3.
Klein, U.R., Nigg, E.A., and Gruneberg, U. (2006). Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP. Mol Biol Cell 17, 2547-2558.
4.
Li, X., Sakashita, G., Matsuzaki, H., Sugimoto, K., Kimura, K., Hanaoka, F., Taniguchi, H., Furukawa, K., and Urano, T. (2004). Direct association with inner centromere protein (INCENP) activates the novel chromosomal passenger protein, Aurora-C. J Biol Chem 279, 47201-47211.
5.
Verhey, K.J., Lizotte, D.L., Abramson, T., Barenboim, L., Schnapp, B.J., and Rapoport, T.A. (1998). Light chain-dependent regulation of Kinesin's interaction with microtubules. J Cell Biol 143, 10531066.
