The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.
The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.
A large number of motility events within eukaryotic cells are mediated by the function of molecular motor proteins. These motors move along the cytoskeletal filaments, actin filaments, and microtubules (MTs), and convert the chemical energy of ATP hydrolysis into kinetic and mechanical forces required to drive biological motility within cells. The MT-based S. cerevisiae Cin8 is a bipolar, homotetrameric kinesin-5 motor protein that crosslinks and slides spindle MTs apart1. Cin8 performs essential functions during mitosis, in spindle assembly2,3,4 and spindle elongation during anaphase5,6,7. Previously, it had been demonstrated that Cin8 is a bi-directional motor, which switches directionality under different experimental conditions. For instance, under high ionic strength conditions, single Cin8 motors move toward the minus-end of the MTs, while in clusters, in multi-motor MT gliding assays, and between antiparallel MTs, Cin8 motors move mainly toward the plus-ends of the MTs8,9,10,11,12. These findings were highly unexpected because of several reasons. First, Cin8 carries its catalytic motor domain at the amino-terminus and such motors were previously believed to be exclusively plus-end directed, whereas Cin8 was shown to be minus-end directed at the single-molecule level. Second, kinesin motors were believed to be unidirectional, either minus-end or plus-end directed, whereas Cin8 was shown to be bi-directional, depending on the experimental conditions. Finally, because of the MT orientation at the mitotic spindle, the classical role of kinesin-5 motors in the separation of spindle poles during spindle assembly and anaphase B could only be explained by their plus-end directed motility on the MTs they crosslink1,13. Following the first reports on the bi-directionality of Cin8, a few other kinesin motors were demonstrated to be bi-directional14,15,16, indicating that the bi-directional motility of kinesin motors may be more common than earlier believed.
It has been previously reported that in cells, Cin8 also moves in a bi-directional manner8, supporting the notion that the bi-directional motility of some kinesin-5 motors is important for their intracellular functions. In addition, since the three kinesin-5 motors that were reported to be bi-directional are from fungal cells, a possible role for the bi-directionality of kinesin-5 motors has been recently proposed in such cells10. According to this model, in closed mitosis of fungal cells, where the nuclear envelope doesn't break down during mitosis, kinesin-5 motors provide the initial force that separates the spindle poles apart prior to spindle assembly. To perform this task, prior to spindle pole separation, kinesin-5 motors localize near the spindle poles, by their minus-end directed motility on single nuclear MTs. Once at this position, kinesin-5 motors cluster, switch directionality, capture, and cross-link MTs from neighboring spindle poles. Subsequently, kinesin-5 motors provide the initial separation of the poles by plus-end directed motility on the MTs they crosslink. By this model, both minus-end directed motility on single MTs and plus-end directed motility on cross-linked MTs during antiparallel sliding are required for fungal kinesin-5 motors to perform their roles in spindle assembly1,13.
The overall goal of the described method is to obtain high-purity fungal GFP-tagged kinesin-5 Cin8 and to perform single-molecule motility assays (Figure 1) while separately analyzing the motility of single molecules and clusters of Cin8. The separation between single molecules and clusters is important since one of the factors that had been demonstrated to affect the directionality of Cin8 is its accumulation in clusters on the MTs10,12. Alternative motility assays, such as the MT surface gliding and MT sliding assays do not provide information regarding the activity of single motor proteins17,18. The robust single-molecule motility assay and analysis methods described here have been successfully applied to characterize different aspects of kinesin-5 motors, Cin8 and Kip110,11,12,14,19,20.
Here, a detailed protocol is presented for Cin8 overexpression and purification, polymerization of MTs, and the single-molecule motility assay. Furthermore, the analyses to differentiate between single molecules and clusters of Cin8, and to determine single motor and cluster velocities by mean displacement (MD) and mean square displacement (MSD) analysis are also described. This protocol aims to help researchers to visualize all the steps of the procedures and assist with troubleshooting this type of assays.
Figure 1: Schematic representation of the single-molecule motility assay. Biotinylated fluorescent MTs are attached to the glass surface, coated with Avidin that interacts with the surface-attached biotinylated-BSA. The green arrow represents the movement direction of single Cin8 molecules under high ionic strength conditions. +/- represent the polarity of the MT. Please click here to view a larger version of this figure.
1. Preparation of buffers and reagents
Volume | Stock | Reagent name |
50 µL | 2X | MB (from step 1.3.1) |
40 µL | – | TDW |
1 µL | 100 mM | ATP |
1 µL | 200 mM | MgCl2 |
2 µL | 5 mg/mL | Casein |
1 µL | 1 M | Glucose |
1 µL | 1 M | DTT |
1 µL | 10 mg/mL | Glucose oxidase |
1 µL | 8 mg/mL | Catalase |
1 µL | 1 M | Phosphocreatine |
1 µL | 5 mg/mL | Creatine phosphokinase |
100 µL | Total |
Table 1.
2. Cin8 overexpression and purification from S. cerevisiae cells
Figure 2: Purification of Cin8-GFP. (A) The size exclusion chromatogram of Ni-NTA purified Cin8-GFP, with continuous GFP fluorescence detection through 488 nm excitation and emission at ~510 nm. The Cin8-GFP tetramer elutes at ~10 mL from the SEC column (marked with an arrow). (B) Coomassie-stained SDS-PAGE gel (top) and α-GFP western blot (bottom) of Cin8-GFP fractions eluted from SEC. Samples in the lanes are as follows: M – Molecular weight marker, Ni2+– Ni-NTA purified Cin8-GFP sample that is loaded into the SEC column, GF fractions: fraction corresponding to Cin8-GFP SEC elution as marked in panel A. The arrow on the right marks the size of the Cin8-GFP monomer (expected on the SDS-PAGE). Please click here to view a larger version of this figure.
3. Single-molecule motility assay with the purified Cin8
Figure 3: MTs and MT bound Cin8-GFP. (A) Images from two fields (left and right) for MTs polymerized following the protocol described in step 3.1 and imaged with 100x objective as described in step 3.4. (B) Images from two fields (left and right) for the Cin8-GFP (lower panels, marked with arrows) attached to the MT shown in the upper panels. Scale bar: 4 µm. Please click here to view a larger version of this figure.
4. Motility analysis
NOTE: Perform all the image analysis and generate kymographs using ImageJ-Fiji Software.
Figure 4: Cin8-GFP bleaching profile and intensity distribution. (A) Photobleaching of GFP in four different Cin8-GFP motors. Single photobleaching steps, each likely representing the photobleaching of one GFP, lead to a drop in fluorescence intensity of ~50 a.u. (B) The intensity distribution of Cin8-GFP motors in the first frame of a time-lapse sequence (inset). The Gaussian peak (blue) centered at ~125 a.u represents single Cin8-GFP molecules. This peak exhibits the average intensity of single Cin8 tetramers with one, two, three, or four fluorescent GFP molecules, with each GFP molecule contributing ~50 a.u. to the total intensity (i.e., (50 + 100 + 150 + 200) / 4 = 125). Please click here to view a larger version of this figure.
The experiment aims to investigate the motility characteristics of bi-directional motor protein Cin8 of different cluster sizes on single MTs. Representative motility of Cin8-GFP is also evident from the kymographs in Figure 5A, where the spatial position of the motor over time is shown.
For the analysis of the motile properties of Cin8-GFP, first, the cluster size is assigned (step 4.3) to each MT-attached motile Cin8-GFP particle, and then the position of the examined Cin8 particles is tracked as a function of time (step 4.4). For each cluster size category, >40 trajectories of individual Cin8-GFP were extracted from the recordings (Figure 5B). Using the coordinates obtained from tracking analysis, MD and MSD analysis is performed for each cluster size population separately. The velocities are obtained from linear fits to MD as presented in Figure 5C. It was found that single Cin8-GFP molecules move in a unidirectional, minus-end directed manner with high velocity, whereas the Cin8 clusters exhibit considerably lower velocity with a higher propensity for bi-directional motility (Figure 5B,C).
Figure 5: Cin8-GFP motility. (A) Kymographs representing motility of Cin8-GFP motors on MTs. X- and Y-axes represent MT lattice and time, respectively. Yellow arrows mark the fast motility of single Cin8-GFP particles toward the minus-end direction of the MT, whereas blue arrows mark the slow motility of Cin8 clusters in the plus-end direction of the MT. The polarity of the MTs is indicated at the bottom of each kymograph (+/-). Horizontal bar: 4 µm, vertical bar: 20 s. (B) Displacement traces of single motors (left) and clusters (right) of Cin8-GFP motors. The displacement traces were plotted using the coordinates obtained after tracking the individual Cin8-GFP motors as explained in step 4.4. Negative and positive values of displacement indicate movement in the minus-end and plus-end directions of the MT, respectively. Note that under the same assay, the motility of Cin8 clusters is slower and bi-directional compared to the single molecules of Cin8. (C) Plots of mean displacement (MD) ± SEM, of single molecules (left) and clusters (right) of Cin8 motors as a function of the time interval. Black lines represent linear fits of the plot (MD = v xt + c, where v is the mean velocity, t is the time interval and c represents the intercept). From the fitting, it is evident that the mean velocity for single motors and clusters of Cin8 is -265 ± 20 nm/s and -48 ± 5 nm/s, respectively. Please click here to view a larger version of this figure.
In this work, a protocol for single-molecule motility assay with the bi-directional kinesin-5 Cin8 and the motility analysis are presented. The full-length Cin818 including the native nuclear localization signal (NLS) at the C-terminal has been purified from the native host S. cerevisiae. As the Cin8 is a nuclear motor protein, grinding the S. cerevisiae cells under liquid nitrogen is found to be the most efficient method for cell lysis. After lysis, by combining metal affinity and size exclusion chromatography, highly pure Cin8 is obtained, which is important for the single-molecule motility assays. It has been previously reported that there are differences between motile properties of Cin8 in crude extracts and purified samples8. In addition, it has also been reported that MT crowding with motor and non-motor proteins affects the directionality of bi-directional kinesin-5 Cut722. Thus, high purity of the motor is required for reliable motility analysis and conclusions regarding wild-type and mutant motor behavior. The techniques described here can be easily adapted to purify other nuclear proteins from the yeast with appropriate buffer adjustments.
Described here is a highly robust and sensitive single-molecule motility assay with GFP-tagged Cin8. The success of this assay relies heavily on the proper MT polymerization and immobilization to the surface. The strong avidin-biotin interaction is utilized to immobilize the MTs to the hydrophobic glass surface, which irreversibly attaches the MTs. On these immobilized MTs using GFP labeled Cin8, Cin8 motility can be reliably tracked11,12,19.
Cin8 is reported to form clusters containing more than one tetrameric motor10,12, with the motility of these clusters being different from that of single Cin8 molecules. To accurately characterize Cin8 motility as a function of its size, a fluorescence intensity-based method has been developed to identify the cluster size of each Cin8 particle12. Based on this size categorization, motility is analyzed separately in each size category. Following this size-based analysis, insightful details are provided, that can be utilized to understand the different behavior of oligomers of the same molecule11,12,19. The cluster size determination procedure described here can be applied to determine the size of a variety of fluorescently labeled molecules. While performing the fluorescence-based size determination, one should be careful to determine the cluster size of Cin8-GFP particles at the first frame of appearance to avoid the impact of bleaching, since the large clusters could appear as smaller ones following photobleaching.
The motility characterization is performed by the MD and/or MSD analyses. If it is of interest to determine only the motor velocity, MD analysis is sufficient. However, if motor motility contains both active and passive components and determination of the diffusion coefficient is also required, MSD analysis should be performed20,23,24,25. For both MD and MSD analyses, the coordinates of the motor for every time point need to be determined. For efficient tracking, it is important to keep the motor concentration optimum. The MTs should not be too crowded with motors; ideally, there should be 3-4 Cin8-GFP motors/particles at a time on an MT of ~10 µm. Automated tools such as the "KymoButler" or "TrackMate" plugin in ImageJ-Fiji can also be used to track the motile motors26,27. These automated tools save time and work, but they have a few limitations. For example, if the motility of some particles is very slow, these tools can read them as non-motile particles. In addition, these tools have limits in recognizing low-intensity molecules. Therefore, they can exhibit a high-intensity bias. On the other hand, manual tracking (although time-consuming) is less sensitive to tracking errors.
In summary, this protocol, starting from the purification of Cin8 overexpressed in S. cerevisiae, explains comprehensively the single-molecule motility assay and the subsequent motility analysis of this bi-directional kinesin-5. This protocol can be followed easily to purify and characterize the motility of motor proteins such as Cin8. Moreover, the different parts of the protocol can be adapted to purify proteins from yeast or develop single-molecule motility assays for different motor proteins and their motility characterization.
The authors have nothing to disclose.
This research was supported in part by the Israel Science Foundation grant (ISF-386/18) and the Israel Binational Science Foundation grant (BSF-2019008), awarded to L.G.
Adenine | FORMEDIUM | DOC0230 | |
ATP | Sigma | A7699 | |
Biotinylated-BSA | Sigma | A8549 | |
Casein | Sigma | C7078 | |
Catalase (C40) | Sigma | C40 | |
Creatine-Kinase | Sigma | C3755 | |
Dithiothreitol (DTT) | Sigma | D0632 | |
EDTA | Sigma | E5134 | |
EGTA | Sigma | E4378 | |
Fluorescence filter set for GFP | Chroma | 49002: ET-EGFP (FITC/Cy2) | |
Fluorescence filter set for Rhodamine | Chroma | 49004: ET-CY3/TRITC | |
Fluorescence inverted microscope | Zeiss | Axiovert 200M | |
Galactose | Tivan Biotech | GAL02 | |
Glucose | Sigma | G8270 | |
Glucose Oxidase | Sigma | G7141 | |
Glycerol | Sigma | G5516 | |
GlycylGlycine | Merck | G0674 | |
GMPCPP | Jana Bioscience | Nu-405L | |
GTB | Cytoskeleton | BST01-010 | |
GTP | Sigma | G8877 | |
Histidine | Duchefa Biochemie | H0710.0100 | |
ImageJ-FIJI software | https://imagej.net/plugins/trackmate/ | version 2.1.0/1.53c; Java 1.8.0_172 [64-bit] for Windows 10 | |
Imidazole | Sigma | I0125 | |
InstantBlue Coomassie Protein Stain | Abcam | ab119211 | |
Lens | Zeiss | 100x/1.4 oil DIC objective | |
Lysine | FORMEDIUM | DOC0161 | |
Magnesium Chloride | Sigma | M8266 | |
Methionine | Duchefa Biochemie | M0715.0100 | |
Neo | Andor Technologies | sCMOS camera | |
NeutraAvidin | Life | A2666 | |
Ni-NTA Agarose | Invitrogen | R901-15 | |
Phospho-Creatine | Sigma | P1937 | |
Pipes | Sigma | P1851 | |
Pluronic acid F-127 (poloxamer) | Sigma | P2443 | |
Potassium Chloride | Sigma | P9541 | |
Raffinose | Tivan Biotech | RAF01 | |
Size Exclusion chromatography instument | GE Healthcare | AKTA Pure | |
Spectrophotometer | ThermoFisher Scientific | NanoDrop | |
Superose-6 10/300 GL | GE Healthcare | 17-5172-01 | |
Tris | Roshe | 10708976001 | |
Triton X-100 | Sigma | T8787 | |
Tryptophan | Duchefa Biochemie | T0720.0100 | |
Tubulin protein | Cytoskeleton | T240 | |
Tubulin, biotinylated | Cytoskeleton | T333P | |
Tubulin, TRITC Rhodamine | Cytoskeleton | TL530M | |
Uracil | Sigma | U0750-100G | |
Yeast nitrogen base | FORMEDIUM | CYN0401S | |
α-GFP antibody | Santa Cruz Biotechnology | SC8036 | |
β-mercaptoethanol | Sigma | M3148 |