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.

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The authors have no conflicts of interest to disclose.

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 &#181;m. Automated tools such as the &#34;KymoButler&#34; or &#34;TrackMate&#34; 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.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63425">Motility of Single Molecules and Clusters of Bi-directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells</a>. The Authors section was updated.
One of the author names was updated from:
Roy Abraham
to:
Roy Avraham

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63425">Motility of Single Molecules and Clusters of Bi-directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells</a>. The Authors section was updated.

Erratum: Motility of Single Molecules and Clusters of Bi-directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

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&#39;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63425/63425fig01v2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63425/63425fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Adenine</td><td>FORMEDIUM</td><td>DOC0230</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>Biotinylated-BSA</td><td>Sigma</td><td>A8549</td><td></td></tr><tr><td>Casein</td><td>Sigma</td><td>C7078</td><td></td></tr><tr><td>Catalase (C40)</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Creatine-Kinase</td><td>Sigma</td><td>C3755</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E4378</td><td></td></tr><tr><td>Fluorescence filter set for GFP</td><td>Chroma</td><td>49002: ET-EGFP (FITC/Cy2)</td><td></td></tr><tr><td>Fluorescence filter set for Rhodamine</td><td>Chroma</td><td>49004: ET-CY3/TRITC</td><td></td></tr><tr><td>Fluorescence inverted microscope</td><td>Zeiss</td><td>Axiovert 200M</td><td></td></tr><tr><td>Galactose</td><td>Tivan Biotech</td><td>GAL02</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Glucose Oxidase</td><td>Sigma</td><td>G7141</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>GlycylGlycine</td><td>Merck</td><td>G0674</td><td></td></tr><tr><td>GMPCPP</td><td>Jana Bioscience</td><td>Nu-405L</td><td></td></tr><tr><td>GTB</td><td>Cytoskeleton</td><td>BST01-010</td><td></td></tr><tr><td>GTP</td><td>Sigma</td><td>G8877</td><td></td></tr><tr><td>Histidine</td><td>Duchefa Biochemie</td><td>H0710.0100</td><td></td></tr><tr><td>ImageJ-FIJI software</td><td>https://imagej.net/plugins/trackmate/</td><td>version 2.1.0/1.53c; Java 1.8.0_172 [64-bit] for Windows 10</td><td></td></tr><tr><td>Imidazole</td><td>Sigma</td><td>I0125</td><td></td></tr><tr><td>InstantBlue Coomassie Protein Stain</td><td>Abcam</td><td>ab119211</td><td></td></tr><tr><td>Lens</td><td>Zeiss</td><td>100x/1.4 oil DIC objective</td><td></td></tr><tr><td>Lysine</td><td>FORMEDIUM</td><td>DOC0161</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Methionine</td><td>Duchefa Biochemie</td><td>M0715.0100</td><td></td></tr><tr><td>Neo</td><td>Andor Technologies</td><td>sCMOS camera</td><td></td></tr><tr><td>NeutraAvidin</td><td>Life</td><td>A2666</td><td></td></tr><tr><td>Ni-NTA Agarose</td><td>Invitrogen</td><td>R901-15</td><td></td></tr><tr><td>Phospho-Creatine</td><td>Sigma</td><td>P1937</td><td></td></tr><tr><td>Pipes</td><td>Sigma</td><td>P1851</td><td></td></tr><tr><td>Pluronic acid F-127 (poloxamer)</td><td>Sigma</td><td>P2443</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Raffinose</td><td>Tivan Biotech</td><td>RAF01</td><td></td></tr><tr><td>Size Exclusion chromatography instument</td><td>GE Healthcare</td><td>AKTA Pure</td><td></td></tr><tr><td>Spectrophotometer</td><td>ThermoFisher Scientific</td><td>NanoDrop</td><td></td></tr><tr><td>Superose-6 10/300 GL</td><td>GE Healthcare</td><td>17-5172-01</td><td></td></tr><tr><td>Tris</td><td>Roshe</td><td>10708976001</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tryptophan</td><td>Duchefa Biochemie</td><td>T0720.0100</td><td></td></tr><tr><td>Tubulin protein</td><td>Cytoskeleton</td><td>T240</td><td></td></tr><tr><td>Tubulin, biotinylated</td><td>Cytoskeleton</td><td>T333P</td><td></td></tr><tr><td>Tubulin, TRITC Rhodamine</td><td>Cytoskeleton</td><td>TL530M</td><td></td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td></td></tr><tr><td>Yeast nitrogen base</td><td>FORMEDIUM</td><td>CYN0401S</td><td></td></tr><tr><td>&amp;alpha;-GFP antibody</td><td>Santa Cruz Biotechnology</td><td>SC8036</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr></tbody></table>

motility of single molecules and clusters of bi-directional kinesin-5 cin8 purified from s. cerevisiae cells

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.

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, &#62;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).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63425/63425fig05.jpg" /> 
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 &#181;m, 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) &#177; 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 &#177; 20 nm/s and -48 &#177; 5 nm/s, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63425/63425fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells at JoVE.com

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

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.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

motility-single-molecules-clusters-bi-directional-kinesin-5-cin8

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.5K Views.  Ben-Gurion University of the Negev, Israel. De Saccharomyces cerevisiae kinesine-5 Cin8 is een bidirectioneel motoreiwit. Cin8 beweegt in de min-eindrichting van de microtubuli als een enkel molecuul en verandert de directionaliteit onder een aantal verschillende experimentele omstandigheden. Het algemene doel van ons onderzoek is om het mechanisme van bidirectionele motiliteit te begrijpen door factoren en structurele elementen te bestuderen die de bidirectionele beweeglijkheid van Cin8 reguleren.Dit protocol verklaart uitgebre...

Video: Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Bioengineering

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

Engineering

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

Biology

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

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