Cloning and protein purification
6.5.1
The construction of the tetrameric chimera DK4mer and a further dimeric deriva-tive was based on a detailed analysis of the transition between motor domain and neck coiled-coil in Eg5 and the known limits of the motor domain and neck linker in the Drosophila Kinesin-1 (DmKHC). In DK4mer the first 345 amino acids (aa) of Drosophila Kinesin-1 were fused to Xenopus Kinesin-5 (Eg5) at aa 370. Additional constructs contained either a 6-his tag (DK4mer-his) or a green fluorescent protein (GFP)-6his cassette as described in detail previously (22). The exact sequence we chose for the transition is schematically shown in Fig. 6.1 A. The dimeric derivative DK511 was truncated at aa 511 (Fig. 6.1 D). Donor plasmids were kindly provided by W. O. Hancock (DmKHC) and T. M. Kapoor (FL-Eg5-GFP, BK006). We used a nested PCR approach similar to that described before [Lak¨amper10] to extend the motor domain and neck linker of DmKHC (DK) with sequences providing a direct and uninterrupted transition using selected restriction sites to a correspondingly am-plified neck/stalk/tail-fragment of Eg5 (EK). DK was amam-plified with a fwd primer, DK1fwd, providing an NdeI site (flanked by additional SalI and XmaI-sequences) and two rev primers, DKrev1 and DKrev2, providing transition sequences of the Eg5 neck up to an AflI site which was generated using a silent mutation in the natural sequence (further flanking regions provide more restrictions sites such as NotI for sub-cloning of fragments). Similarly the EK fragment was generated using a fwd-primer, EKfwd1, and two rev-primers, EKrev1 and EKrev2, providing the same restriction sites at the N-terminus and a cassette containing a sequence containing an AscI and XmaI site followed by a 6his box and a stop codon followed by the cut-ting sides NotI, Sal and XhoI. The AscI/XmaI site allowed us to insert a previously used GFP-6his casette from pT7-7-GFP-his. The resulting PCR-fragments were ini-tially “parked” in a pTOPO-XL vector (Invitrogen, USA), before sub-cloning into a pFastBac vector (Invitrogen, USA) for expression in Sf9-cells. Truncation constructs (DK511-GFPhis as a truncated version of DK4mer and D421-GFPhis as a truncated version of DmKHC) were generated using a simple PCR approach and subsequent sub-cloning in a pET-21b(+) vector (Novagen, Germany). Expression and purifica-tion was performed as described in [Lak¨amper10]. Chemicals were purchased from Sigma, USA, if not otherwise stated.
Multi-motor surface-gliding assays
6.5.2
Multi-motor surface-gliding assays were performed at 22¶C as described previously [Lak¨amper10]. The motor proteins were allowed to non-specifically bind to the glass surfaces of assay chambers made from cover slips and microscope slides, assembled with double-stick tape. Subsequently, the chambers were flushed with about three chamber volumes of assay mix (AM) based on BRB80 buffer (80 mM PIPES/KOH,
92 Chapter 6. A chimeric kinesin-1/kinesin-5 microtubule-sliding motor. . .
pH 6.8, 1 mM MgCl2, 1 mM EGTA) containing 10µm taxol (paclitaxel), 2 mM ATP, 4 mM MgCl2, 10 mM DTT, 0.08 mg/ml catalase C40, 0.1 mg/ml glucose oxidase and 10 mM glucose. For multi-motor surface-gliding assays, 0.022 mg/ml tetramethyl-rhodamine (TMR)-labeled MTs, polymerized as described previously [Lak¨amper10], were added to AM. Motility was observed in a standard inverted fluorescence micro-scope (Zeiss Axiovert 200, Germany) using a Zeiss EC Plan-Neofluar 100◊ 1.3 NA oil immersion objective (Zeiss, Germany). Images were recorded with a digital CCD camera (CoolSnap ES, Roper Scientific, Germany) at a frame rate of 2 frames/s.
Videos were analyzed with ImageJ (National Institute of Health, USA) to obtain MT velocities.
Single-molecule fluorescence assays
6.5.3
Cover slips were cleaned using a plasma cleaner (PDC-002, Harrick Plasma, USA) before surface silanization with 3-[2-(2-Aminoethylamino)ethylamino]propyltrimeth-oxysilane (DETA) (Sigma, Germany) for MT immobilization as described before [Lak¨amper10]. TMR-labeled MTs were allowed to bind to the surface during an incubation time of 5 min., followed by 5 min. incubation with 0.1 mg/ml casein in BRB80. Finally DK4mer-GFPhis (DK4mer) diluted in AM to single-molecule con-centration (≥ 150 nM) was introduced, and fluorescence was observed in a custom-built total-internal-reflection-fluorescence (TIRF) microscope.
The TIRF setup was built similarly to the setup described previously by van Dijket al. [Dijk07] with the following modifications: we used two lasers (473 nm and 532 nm, both Viasho, USA) to excite the fluorophores (GFP and TMR). The lasers were expanded and coupled via a multi-wave-length beam splitter (z474/488/532/635rpc, Chroma, USA) off-axis into an oilimmersion objective (Nikon, SFluor 100◊, 1.49) to obtain TIRF illumination. The emitted fluorescent light was split in the GFP and TMR signals using the dichroic mirror (525/50, Chroma, USA), then passed trough bandpass filters (530/50 for GFP and 605/70 for TMR, both Chroma, USA) and finally directed via mirrors to separate areas of the detector area of a frame-transfer EMCCD camera (Cascade 512B, Roper Scientific, USA). In a further modification (not used for the experiments in this work) a commercial image splitter (Optosplit III, Cairn Research, UK) was integrated into the setup (Supplementary Fig. D.4) providing the possibility to detect an additional, third fluorophore. For analysis, the separated signals of GFP and TMR were superimposed using the OptoSplit ImageJ plugin provided by Cairn Research, UK.
For measurements at different salt concentrations, KCl was added to BRB80 buffer. Measurements with lower than 80 mM salt concentration were done in AM as described above, but based on P30 buffer (30 mM PIPES/KOH, pH 6.8, 1 mM MgCl2, 1 mM EGTA). To calculate the total ionic strength of the different buffers, one has to take into account that PIPES buffer is a diprotonic acid (pKa1 < 3, pKa2 = 6.8). To adjust, e.g., 80 mM of PIPES free acid to pH 6.8, the addition of 120 mM KOH is required. At pH 6.8, additional to the K+ and Cl≠ ions, an equimolar amount of each acid group is in solution, and the OH≠ ions are buffered
Section 6.5. Materials and Methods 93
by water. Ionic strength can be calculated as: I = 12qciz2i, with ci concentration and zi valence of ion type i. BRB80 plus 10 mM KCl, for example, has an ionic strength I = 1/2 (40◊ 1 + 40 ◊4 + (120 + 10) ◊ 1 + 10 ◊ 1) mM = 170 mM.
All single-molecule-fluorescence measurements were performed at 22 ¶C. The CCD camera was controlled with WinSpec32 software (Princeton Instruments, USA).
Digital images were recorded at a frame rate of 2 frames/s and were subsequently analyzed for velocities and run lengths using kymographs generated with a custom-written LabView (National Instruments, USA) routine. To estimate average ve-locities, only the straight (judged by eye) runs or straight segments of runs were taken into account. Runs lasting for less then 2 seconds were not scored. Statisti-cal analysis of data was performed with OriginPro (OriginLab Corporation, USA).
MSD analysis was done with a custom-written MATLAB (The MathWorks, USA) routine.
Relative sliding of polarity-marked MTs
6.5.4
Polarity-marked MTs were assembled in two polymerization steps: First, highly con-centrated TMR-labeled tubulin (3.33 mg/ml) was polymerized in the presence of 0.4 mM guanosine-5’-[(–,—)-methyleno]triphosphate (GMP-CPP, Jena Bioscience, Ger-many) to avoid depolymerization after dilution. In the second step, these short MTs (seeds) were diluted and extended by further polymerization in a solution of 0.54 mg/ml TMR-labeled tubulin and 0.72 mM GTP in P30 buffer. Additionally to a batch of long polarity-marked MTs, a batch of short polarity-marked MTs was poly-merized by increasing the seed concentration and shortening the incubation time.
Finally the MTs were stabilized in P30 buffer containing 10 µM taxol (paclitaxel, Sigma, USA). DETA-silanized assay chambers were prepared as described above.
Long MTs were allowed to bind for≥ 4 min., followed by≥6 min. incubation with 0.5 mg/ml casein in P30 buffer. Finally, 10µl of AM based on P30 buffer containing 4 - 5 times the single-molecule concentration (1:150 dilution) of DK4mer and short MTs were added into the flow chamber.
For the measurements at different salt concentrations, AMs based on P12 buffer (12 mM PIPES/KOH, 1 mM MgCl2, 1 mM EGTA), P30 buffer and BRB80 buffer were used. Fluorescence was observed with a wide-field fluorescence microscope (Zeiss Axiovert 200, Germany) equipped with a Zeiss EC Plan-Neofluar 100x 1.3 NA oil immersion objective. Images were recorded with a CCD camera (Coolsnap ES, Roper Scientific, Germany). All measurements were performed at 22 ¶C and videos were recorded at a frame rate of 2 frames/s. Digital images were analyzed using kymographs generated with custom-written LabView (National Instruments, USA) routines for MT velocity and binding geometry.
Single-molecule imaging of motors during MT sliding
6.5.5
Single motor molecules were imaged during relative sliding of polarity-marked MTs in the custom-built TIRF setup. The assay was performed similarly to the
(multi-94 Chapter 6. A chimeric kinesin-1/kinesin-5 microtubule-sliding motor. . .
motor) relative sliding assay of polarity-marked MTs, but with a 1:280 dilution of the motor protein DK4mer. The measurements were performed at 22 ¶C, and videos were recorded at a frame rate of 2 frames/s. The TMR- and the GFP-signals, recorded on separate areas of the CCD detector, were aligned using the OptoSplit ImageJ plugin provided by Cairn Research, UK and analyzed for motor protein and MT velocity as described above.
Acknowledgements 6.6
This work was supported in part by the Lower Saxony Grant no. 11-76251-99-26/08 (ZN2440) and in part by the Center for Molecular Physiology of the Brain (CMPB), funded by the Deutsche Forschungsgemeinschaft (DFG). We thank Marcel Bremerich for programming the MSD analysis routine.
Supplementary data
Supplementary data associated with this manuscript can be found in appendix D.