Cloning and protein purification
4.4.1
The full-length Eg5Kin chimera was constructed from pPK113 pET5a-FL DmKHC-His) [Coy99a] and pBK006 (FL-Eg5-GFP) [Kwok06] using a nested-PCR approach in order to extend the sequence of the Eg5 motor domain (1 - 369) with the DmKHC residues starting at 345 towards an existing HindIII restriction site in pPK113. The DmKHC motor domain in pPK113 was subsequently replaced by sub-cloning of this transition clone using NdeI and HindIII. A shortened GFP-tagged clone was truncated at residues corresponding to DmKHC 421, using PCR primers provid-ing an AscI and XmaI site upstream of a stop-codon and replaced the full-length DmKHC in pPK113 using NdeI and NotI. The truncated and his-tagged wild-type (WT) Eg5 constructs 369 and 511 were generated using primers providing the same flanking restriction sites for insertion into pPK113. A GFP-6His cassette flanked by AscI and XmaI was generated in pT7-7 for insertion downstream of the truncated Eg5Kin construct and the truncated WT constructs in pPK113. Integrity was con-firmed by sequencing and expression and purification was performed essentially as previously described in [Coy99a]. In brief, BL21(DE3) cells (Invitrogen, CA, USA) were transformed and grown to a density of ≥ 0.6 at 37 ¶C before induction of expression using 1 mM IPTG for 3 h. Cells were harvested by centrifugation and resuspended in a 20 mM imidazole buffer (pH 7.4), supplemented with 1 mM each of DTT, MgCl2, EGTA, BME and 150 mM NaCl before lysis using ultrasound in the presence of lysozyme and DNAseI. After separation from cell debris by centrifu-gation, the cytosol was incubated for 1 h at 4 ¶C with Ni-NTA-column material (Qiagen, Germany) and then transferred to a syringe column. After washing the column with 80 mM imidazol and 300 mM NaCl, bound motor was eluted using a 300 mM imidazol buffer containing 10 µm ATP and 1 mM DTT. Fractions contain-ing motor were pooled and 3◊ dialyzed against 80 mM PIPES/KOH, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 µM ATP. The truncated motors were purified using a MT-affinity purification procedure described in [Lak¨amper05]. Protein samples were tested for motor activity and stored in aliquots at - 80 ¶C. Microtubules were purified from pig brain and labeled as described in [Lak¨amper05].
Surface-gliding assays
4.4.2
Surface gliding assays were performed at 21 ¶C essentially as described previously [Lak¨amper05]. Full-length Eg5Kin motors were allowed to non-specifically bind to the glass surfaces of assay chambers made from cover-slips and microscope slides as-sembled with double-stick tape. Subsequently, the chambers were flushed with about three chamber volumes of assay mix in BRB80 buffer (80 mM PIPES/KOH, 1 mM MgCl2, 1 mM EGTA) containing 10 µm taxol (paclitaxel), 0.022 mg/ml tetram-ethylrhodamine (TMR)-labeled microtubules, 2 mM ATP, 4 mM MgCl2, 10 mM
Section 4.4. Materials and Methods 63
DTT, 0.08 mg/ml catalase, 0.1 mg/ml glucose oxidase and 10 mM glucose. Motility was observed in a standard inverted fluorescence microscope (Zeiss Axiovert 100, Germany). In surface-gliding assays in the presence of up to 1250 µM monastrol, only moving microtubules were scored to obtain average velocities. A small fraction of microtubules got irreversibly stuck on surface contaminations in the absence of monastrol. Above≥260µM monastrol, no moving microtubules were observed any-more. Images were recorded with a digital CCD camera (CoolSnap ES, Roper Sci-entific, Germany). The same protocols were used for the truncated motor construct, Eg5Kin-GFP and for non-chimeric Eg5 truncation constructs Eg5-369(GFP)His and Eg5-511(GFP)His. Alternatively, motors were introduced in chambers pre-treated with streptavidin and biotinylated penta-His antibody (Qiagen, Hilden, Germany).
Single-molecule optical-trapping assays
4.4.3
Assays were performed at 21 ¶C using a single-beam optical trap setup built on a custom-designed inverted microscope as described elsewhere [Korneev07]. Infrared laser light (1064 nm, cw, Nd:YVO4, Compass, Coherent, Santa Clara, USA) was focused into the flow chamber using an objective (Zeiss Neofluar 100◊ oil, 1.3 NA, G¨ottingen, Germany) to form an optical trap. Trap stiffness was varied in the range of 3 - 6 ◊ 10≠5 N/m. The back-focal plane of the condenser (Zeiss, oil, 1.4 NA) was imaged onto a quadrant photodiode which was operated at a reverse bias of 100 V (YAG-444-4A, Perkin-Elmer, Vaudreuil, Canada) for position detection of the trapped particle. Photo diode signals that correspond to the displacement fluctua-tions of the trapped particle in the plane normal to the optical axis were processed by custom-built analog electronics [Allersma98] and read into a PC using LabView (National Instruments, Austin, TX, USA). For motility assays, streptavidin-coated silica spheres (0.5 µm diameter, Kisker Biotech, Steinfurt, Germany) were incu-bated with 0.1 mg/ml casein in PEM12 buffer (12 mM PIPES/KOH, 2 mM MgCl2, 1 mM EGTA, pH 6.8) prior to incubation with biotinylated antibodies against the GFP tag (Goat Anti-GFP polyclonal antibody, biotin conjugated, Abcam plc, Cam-bridge, UK) for 30 min. Beads were washed at least three times in PEM12 containing 0.1 mg/ml casein before dilute motors were added in the presence of 1 mM ATP.
Microtubules were added into silanized flow chambers (see below) and allowed to bind to the surface for 5 min before blocking the surface with 0.1 mg/ml casein in PEM12. An assay mix containing motor-covered beads in PEM12 with added 10 mM dithiothreitol (DTT), 1 mM ATP, 2 mM MgCl2, 10 mM glucose, 100µg/ml glu-cose oxidase, 80 µg/ml catalase, 0.1 mg/ml casein was flushed into the pre-treated chamber, and motility was observed as described above. To ensure that beads car-ried only one functional motor, the motor concentration was reduced such that only about one in ten beads produced motility.
64 Chapter 4. The effect of monastrol on the processive motility of a dimeric. . .
Single-molecule fluorescence processivity assays
4.4.4
Tetramethyl-rhodamine labeled microtubules were polymerized in the presence of guanosine-5’-[(–,—)-methyleno]triphosphate (GMP-CPP) to avoid de-polymerization after dilution. Microtubules were attached to positively charged silanized cover slips. Cover slips were cleaned using KOH/EtOH before surface silanization using 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (DETA) (Sigma, Ger-many) for microtubule immobilization. Microtubules were allowed to bind for 5 min followed by 5 min incubation with 0.1 mg/ml BSA or casein in BRB80. A 1:20 000 dilution of Eg5Kin-GFP in assay mix was introduced, and fluorescence was observed in a custom-built wide-field fluorescence microscope [Kapitein05]. For assays including monastrol, the assay mix contained at most 5 % DMSO. Digital images were subsequently analyzed for speeds, run lengths, binding rates and asso-ciation times using kymographs generated with custom-written LabView software.
Statistical analysis of data was performed with OriginPro (OriginLab Corporation, Northampton, USA).
Acknowledgements 4.5
We thank J. van Mameren, M. Noom, and S. van den Wildenberg for software devel-opment and help, and W. O. Hancock, and B. Kwok/T. Kapoor for the generous gifts of the plasmids pPK113 and BK006, respectively. This work was partially supported by a research grant from the Human Frontiers Science Program (S.L. and C.F.S.).
L.C.K. and E.J.G.P. were supported by a VIDI grant from the Dutch Organization for Scientific Research (NWO) and a travel grant from the Laser Center of the Free University Amsterdam. The work was further supported by the Research Center for Molecular Physiology of the Brain, funded by the Deutsche Forschungsgemeinschaft.
Supplementary data
Supplementary data associated with this article can be found online at the Journal of Molecular Biology, doi:10.1016/ j.jmb.2010.03.009and in appendix B.