Name: single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors
Uploaded: 2022-07-27T13:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

V.S. and P.S. thank Prof. Kristen J. Verhey (University of Michigan, Ann Arbor, MI, USA) and Prof. Roop Mallik (Indian Institute of Technology Bombay (IITB), Mumbai, India) for their unconditional support throughout the study. P.S. thanks Dr. Sivapriya Kirubakaran for her support throughout the project. V.S. acknowledges funding through DBT (Grant No.: BT/PR15214/BRB/10/1449/2015 and BT/RLF/Re-entry/45/2015) and DST-SERB (Grant No.: ECR/2016/000913). P.K.N acknowledges ICMR for funding (Grant No. 5/13/13/2019/NCD-III). P.S. acknowledges funding from DST (Grant No.: SR/WOS-A/LS-73/2017). D.J.S acknowledges&#160;fellowship from IIT Gandhinagar.

1. Sf9 culture, transfection, and virus generation
NOTE: Maintain Sf9 cells in 30 mL of Sf-900/SFM medium in 100 mL sterile, disposable conical flask without any antibiotic/antimycotic at 28 &#176;C. Keep the suspension culture in an orbital shaker at 90 rpm. Supply of CO2 and humidity maintenance is not required. Cells are usually subcultured every fourth day by inoculating 0.5 x 106 cells/mL to reach 2.0 x 106 cells/mL density on the fourth day.
<ol>
	<li>P0 virus stock generation
	<ol>
		<li>For transfection, seed cells into a 35 mm dish with 4.5 x 105 cells/mL confluency and maintain at 28 &#176;C without shaking.</li>
		<li>After 24 h, once cells are attached and look healthy, proceed for transfection as described below.
		<ol>
			<li>Tube A: Mix 1 &#181;g of bacmid DNA encoding for constitutively active KIF1A(1-393LZ)-3xmCit-FLAG specific kinesin-3 motor with 100 &#181;L of unsupplemented Grace&#39;s media.</li>
			<li>Tube B: Mix 6 &#181;L of transfection reagent with 100 &#181;L of unsupplemented Grace&#39;s media.</li>
			<li>Carefully transfer the content of Tube A into Tube B and mix thoroughly by pipetting up and down (approximately 20 times).</li>
			<li>Incubate the mixture for ~45 min at room temperature.</li>
		</ol>
		</li>
		<li>After completing the incubation, add 0.8 mL of unsupplemented Grace&#39;s media to the above mixture and mix slowly by pipetting.</li>
		<li>Gently aspirate the Sf-900/SFM media from cells (to remove any traces of serum that may affect transfection efficiency).</li>
		<li>Add the transfection mixture from step 1.1.3 dropwise on the top of the cells and incubate the plate for 6 h at 28 &#176;C.</li>
		<li>After the incubation, carefully remove the transfection mixture, add 2 mL of Sf-900/SFM media and incubate further for 48 h at 28 &#176;C.</li>
		<li>Check for the motor protein expression under an inverted fluorescence microscope. The motor protein is tagged with a fluorescent protein, mCitrine, a variant of yellow fluorescent protein. 
		NOTE: Transfected cells were visualized under an inverted microscope equipped with differential interference contrast (DIC) and epifluorescence illumination with a 20x objective (200 times magnification), mercury lamp and an EM-CCD camera. Check the efficiency of virus generation and infection by monitoring the expression of mCitrine-tagged motors in cells through mCitrine excitation and emission filter cube. In addition, check for morphological changes of infected cells, such as enlarged cells/nuclei (Figure 1A-C).</li>
		<li>Again, check the cells 72 h post-infection. Usually, cells start detaching from the surface (Figure 2).</li>
		<li>If &#62;5% of cells detached from the surface, harvest the media with infected cells in 1.5 mL sterile microcentrifuge tubes and spin for 5 min at 500 x g.</li>
		<li>Collect the supernatant and snap freeze aliquots of 1 mL in liquid nitrogen and store as P0 stock at -80 &#176;C or use it to generate P1 virus stock.</li>
	</ol>
	</li>
	<li>P1 virus stock generation
	<ol>
		<li>To further amplify the P0 baculovirus stock and confirm protein expression, grow Sf9 cells in liquid suspension culture.</li>
		<li>In a sterile 100 mL conical flask, add 10 mL of Sf-900/SFM media with a cell density of 2 x 106 cells/mL and 1 mL of P0 virus stock. Incubate at 28 &#176;C with constant shaking at 90 rpm.</li>
		<li>After 72 h of infection, check for the protein expression as described earlier (step 1.1.7). If the protein expression is good (&#62;90% of cells show a bright mCitrine fluorescence signal) and shows significant cell death (approximately 10%-15%), spin down the cells in a 15 mL sterile conical tube at 500 x g for 5 min.</li>
		<li>Collect the supernatant, snap freeze aliquots of 1 mL (P1 stock) in liquid nitrogen and store at -80&#176;C or proceed for large-scale infection and protein purification.</li>
	</ol>
	</li>
	<li>Large-scale infection
	<ol>
		<li>For large-scale protein expression, infect 30 mL of the suspension culture at 2 x 106 cells/mL density with 1 mL of P1 virus stock and incubate at 28 &#176;C with constant shaking at 90 rpm.</li>
		<li>After ~72 h post-infection, check for the protein expression (in general, maximum protein expression is achieved between 65-75h post-infection).</li>
		<li>If &#62;90% of cells show a bright fluorescent signal with minimal cell death (&#60;5%), collect the cells in a sterile 50 mL conical tube and spin down at 500 x g at 4 &#176;C for 15 min. 
		&#8203;NOTE: There should be minimal cell death (&#60;5%), because dead cells release the cellular contents into the media, which leads to loss of expressed protein.</li>
		<li>Discard the supernatant, collect the cell pellet, and proceed with protein purification.</li>
	</ol>
	</li>
</ol>
2. Sf9 purification of kinesin-3 motors
<ol>
	<li>To the above cell pellet, add 3 mL of ice-cold lysis buffer (Supplemental Table 1) freshly supplemented with 5 mM DTT, 5 &#181;g/mL of aprotinin, 5 &#181;g/mL of leupeptin, and 5 &#181;g/mL of PMSF and lyse the cells by pipetting 20-25 times without generating any air bubbles. For all the buffer compositions and reagents, please refer to Supplemental Table 1.</li>
	<li>Spin the cell lysate at 150,000 x g for 30 min at 4 &#176;C.</li>
	<li>Collect the supernatant into a fresh, sterile tube and mix with ~40 &#181;L of 50% anti-FLAG M2 affinity resin. Incubate the mixture for 3 h at 4 &#176;C with end-to-end tumbling.</li>
	<li>After incubation, pellet the FLAG resin by spinning at 500 x g for 1 min at 4 &#176;C. Gently aspirate the supernatant without disturbing the pellet with a 26 G needle and discard.</li>
	<li>Wash the FLAG resin pellet three times with ice-cold wash buffer (Supplemental Table 1) freshly supplemented with 2 mM DTT, 5 &#181;g/mL of aprotinin, 5 &#181;g/mL of leupeptin, and 5 &#181;g/mL of PMSF. Pellet the beads by spinning at 500 x g for 1 min at 4 &#176;C in each wash.</li>
	<li>After the third wash, carefully drain the wash buffer as much as possible without disturbing the pellet. For protein elution, add ~70 &#181;L of wash buffer containing 100 &#181;g/mL FLAG peptide to the resin pellet and incubate overnight at 4 &#176;C with end-to-end tumbling.</li>
	<li>On the subsequent day, spin down the resin at 500 x g for 1 min at 4 &#176;C. Collect the supernatant containing purified protein into a fresh tube and supplement with 10% glycerol. Snap freeze aliquots of 5 &#181;L in liquid nitrogen and store at -80 &#176;C until further use.</li>
	<li>Run the SDS-PAGE gel to determine the protein concentration and yield. Along with purified protein of interest, a standard protein control, BSA of known concentrations ranging from 0.2 &#181;g, 0.4 &#181;g, 0.6 &#181;g, 0.8 &#181;g, and 1 &#181;g are loaded to generate a standard curve. Stain the gel with Coomassie Brilliant blue (Figure 3).</li>
	<li>Analyze the gel using a built-in gel quantification tool in ImageJ software. First, measure the band intensity of known concentrations of BSA and generate the standard curve. Then, measure the intensity of the purified protein band and determine the protein concentration. To do so open the Image J software, click on the Analyze option in the menu bar and select Gels in the drop-down menu.</li>
</ol>
3. In vitro single-molecule motility assay using Sf9-purified kinesin-3 motors
NOTE: The Sf9-purified kinesin-3 motors can be used to study biochemical and biophysical properties such as ATP turnover rate, microtubule affinity, velocity, run length, step size, and force generation. Here, a detailed protocol for in vitro single-molecule motility analysis of KIF1A(1-393LZ) using Total Internal Reflection Fluorescence (TIRF) microscopy is described. For all the buffers composition and reagents, please refer to Supplemental Table 1.
<ol>
	<li>Microtubule polymerization
	<ol>
		<li>In a pre-chilled 0.5 mL microcentrifuge tube, prepare a polymerization mix by pipetting in the following order: 12.0 &#181;L of BRB80 buffer, pH 6.9; 0.45 &#181;L of 100 mM MgCl2, 1 &#181;L of 25 mM GTP.</li>
		<li>Take out a 10 &#181;L aliquot of 10 mg/mL tubulin stored in liquid nitrogen, thaw immediately, and add into the above polymerization mixture. Mix gently by pipetting 2-3 times without creating any air bubbles. 
		NOTE: Perform the above steps quickly and strictly on ice.</li>
		<li>Let the above mixture sit on ice for 5 min. 
		NOTE: This is a critical step to prevent denaturing of tubulin or to avoid the formation of short microtubule seeds.</li>
		<li>Transfer the tube into a pre-warmed 37 &#176;C heat block/water bath and incubate for 30 min for polymerization of microtubules.</li>
		<li>While microtubules are polymerizing, thaw an aliquot of P12 buffer and bring it to room temperature.</li>
		<li>Before completing 30 min of incubation, start preparing MT stabilization buffer by pipetting 100 &#181;L of P12 buffer into a fresh microcentrifuge tube. To this, add 1 &#181;L of 1 mM taxol and immediately vortex the mixture. 
		NOTE: Start preparing the MT stabilization buffer approximately 5 min before completing the incubation in step 3.1.4. Taxol stabilizes the polymerized microtubules by binding to &#946;-tubulin.</li>
		<li>Warm the microtubule stabilization buffer for 2-3 min at 37 &#176;C and gently add it to the polymerized microtubules without disturbing the polymerization mixture at the bottom. 
		&#8203;NOTE: Warming the stabilization buffer will bring it to the same temperature as the polymerization mixture.</li>
		<li>Do not tap or pipette the mixture and incubate further at 37 &#176;C for 5 min.</li>
		<li>Gently tap the mixture and mix it with a beveled cut tip (200 &#181;L capacity) slowly using the pipette. 
		NOTE: From this point onward, always handle microtubules with a beveled cut tip to avoid microtubule shearing.</li>
		<li>Take 10-15 &#181;L of polymerized microtubules in a flow cell to check proper microtubule polymerization. 
		NOTE: One can visualize the unlabeled microtubules using a DIC microscopy setup.</li>
		<li>If microtubules are concentrated, dilute them in P12 buffer supplemented with 10 &#181;M taxol.</li>
	</ol>
	</li>
	<li>Preparation of motility flow cell chamber
	<ol>
		<li>Prepare a motility chamber using a glass slide, double-sided tape and glass coverslip (22 mm x 30 mm).</li>
		<li>Take a glass slide, place a drop (~70 &#181;L) of deionized distilled water in the middle and wipe it with a lint-free tissue paper.</li>
		<li>Cut two strips of double-sided tape (~35 x 3 mm) and firmly stick them to the glass slide parallelly, leaving an ~4-5 mm gap between two strips to create a narrow passage.</li>
		<li>Next, take a coverslip and add a drop (~20 &#181;L) of deionized distilled water in the middle. Place a strip of lint-free lens cleaning tissue paper on the water drop until it absorbs the water. Then, slide it slowly toward one end of the coverslip. 
		NOTE: The coverslip should be completely dry. No water should be visible on the coverslip.</li>
		<li>Place the coverslip on the double-sided strips stuck on the slide and press the coverslip evenly along the strips to stick firmly. 
		NOTE: Please make sure that the cleaned side of the coverslip faces the glass slide.</li>
		<li>Ensure that together this creates a narrow chamber of 10-15 &#181;L capacity for performing motility assay (Figure 4A).</li>
	</ol>
	</li>
	<li>In vitro single-molecule motility assay 
	NOTE: To study the microtubule-based single-molecule motility properties of motors, microtubules need to be adsorbed onto the coverslip surface in the motility chamber.
	<ol>
		<li>Dilute the polymerized taxol-stabilized microtubules in P12 buffer supplemented with 10 &#181;M taxol at 1:5 ratios and mix by pipetting slowly with a beveled tip.</li>
		<li>Keep the flow chamber in a slant position (~15-20&#176;). Flow 30 &#181;L of diluted microtubule solution through the flow chamber from the upper end while keeping a lint-free tissue paper at the lower end to absorb the liquid. This creates a shear force to align the microtubule in the flow direction and helps to adsorb the microtubules straight and align parallel.</li>
		<li>Leave a small drop of liquid on both ends of the chamber and keep the flow cell in an inverted position (coverslip facing the bottom) in a closed, moist chamber to prevent drying of the motility chamber.</li>
		<li>Let it sit for ~30 min so that microtubules adsorb to the surface of the coverslip inside the motility chamber.</li>
		<li>In the meantime, prepare&#160;blocking buffer by mixing 500 &#181;L P12-BSA buffer with 5 &#181;L of 1 mM taxol.</li>
		<li>Flow 40-50 &#181;L of blocking buffer and incubate the slide in an inverted position for 10 min in a moist chamber.</li>
		<li>Prepare the&#160;motility mixture by pipetting the following components into a 500 &#181;L capacity sterile microcentrifuge tube in the following order: 25 &#181;L of P12 buffer with taxol, 0.5 &#181;L of 100 mM MgCl2, 0.5 &#181;L of 100 mM DTT, 0.5 &#181;L of 20 mg/mL Glucose oxidase, 0.5 &#181;L of 8 mg/mL Catalase, 0.5 &#181;L of 2.25 M Glucose, and 1.0 &#181;L of 100 mM ATP. 
		NOTE: Fluorescence imaging has been widely used for biological applications22,23,24,25,26. Photoexcitation of fluorescent proteins generates reactive oxygen species (ROS), which can cause photobleaching of the fluorescent proteins and damage to the biological samples27,28. Oxygen scavengers such as glucose, glucose oxidase and catalase are routinely used in motility assays to limit photodamage and prolong the bleaching time of fluorescent proteins.</li>
		<li>Finally, add 1 &#181;L of the purified motor to the above motility mix and mix well before flowing into the motility chamber.</li>
		<li>Seal both the ends of the motility chamber with liquid paraffin wax and immediately image under TIRF illumination using 100X TIRF objective of 1.49 NA with 1.5x magnification.</li>
		<li>In order to focus the coverslip surface, first, focus on one of the inner edges of double-sided tape in the motility chamber under differential interference contrast (DIC) illumination. 
		NOTE: The bright and uneven surface will be visible.</li>
		<li>Then, move the focus into the motility chamber. Using fine adjustment, focus the coverslip surface and look for the microtubules adsorbed on the coverslip surface.</li>
		<li>Once the microtubules are focused, switch to TIRF illumination with a 488 nm excitation laser and adjust the illumination depth by changing the excitation beam angle to get the best and uniform TIRF illumination (Figure 4B).</li>
		<li>Focus the individual mCitrine-tagged motors moving processively along the microtubule surface with 100 ms exposure and record the motion using an EM-CCD camera. 
		NOTE: Although the motility assays were performed on unlabeled microtubules, the maximum intensity z-projection function can be used to reveal the outline of the microtubule track. Preferentially, events on long microtubule tracks were considered for tracking analysis.</li>
		<li>Manually track the position of the fluorescently tagged individual motors walking on long microtubule tracks frame-by-frame using a custom-written plugin in ImageJ (nih.gov) software as described previously29.</li>
		<li>Generate the histograms of velocity and run length for the motor population by plotting the number of events in each bin. Fit these histograms to a single Gaussian peak function to obtain average velocity and run length12 (Figure 4C-E).</li>
	</ol>
	</li>
</ol>
4. In vitro microtubule gliding assay
NOTE: To understand the collective behavior of kinesin-3 motors, in vitro microtubule gliding assay was performed18,30,31. Where motors are immobilized onto the coverslip in an inverted position and upon adding microtubules into the chamber, microtubules land on motors and glide along as the motors try to walk on them (Figure 5A,B).
<ol>
	<li>Fluorescent microtubule polymerization:&#160;Polymerize the microtubules following the protocol described previously except for mixing rhodamine labeled tubulin (3 mg/mL) with unlabeled tubulin (10 mg/mL) in a ratio 1:10.</li>
	<li>After 30 min of polymerization, gently add 30 &#181;L of pre-warmed microtubule stabilization buffer and incubate further for 5 min at 37 &#176;C.</li>
	<li>Next, shear the microtubules by pipetting with the capillary-loading tip (~25-30 times).</li>
	<li>Prepare the motility flow chamber as described previously, flow 50 &#181;L of Sf9-purified GFP nanobodies (2.5 &#181;L of 100 nM diluted in 50 &#181;L of P12 buffer) and incubate for 30 min at room temperature with the coverslip facing downward in a moist chamber. 
	NOTE: The motors are C-terminally tagged with mCitrine. GFP nanobodies are used to immobilize the motors due to their low dissociation constant32.</li>
	<li>Block the coverslip surface by flowing 50 &#181;L of block buffer into the flow chamber to prevent nonspecific protein adsorption and incubate further for 5 min.</li>
	<li>Prepare the motor mix by pipetting 50 &#181;L of block buffer, 1 &#181;L of 100mM ATP, and 5 &#181;L of 100 nM Sf9-purified kinesin-3 motors. Mix gently before flowing into the chamber and incubate for 30 min at room temperature in a moist chamber.</li>
	<li>Wash the chamber twice with 50 &#181;L of P12 casein.</li>
	<li>In a sterile microcentrifuge tube, prepare the gliding assay mixture in the following order, 45 &#181;L of P12-casein with 10 &#181;M taxol, 1 &#181;L of 100 mM ATP, 0.5 &#181;L of 8 mg/mL catalase, 0.5 &#181;L of 20 mg/mL glucose oxidase, 0.5 &#181;L of 2.25 M glucose and 1 &#181;L of sheared fluorescent microtubules. Gently mix the content before flowing into the motility chamber and seal the ends of the chamber with liquid paraffin wax.</li>
	<li>Image microtubule gliding under TIRF illumination at 100 ms exposure and capture the images with attached EM-CCD camera. 
	NOTE: The average microtubule gliding velocity was determined by manually tracking approximately 100 individual microtubules frame-by-frame using a custom-written plugin in ImageJ29. Determine the average microtubule gliding velocity by generating a histogram and fitting to a Gaussian function (Figure 5C,D).</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+system:+A+commercial+manufacturing+platform+for+viral+vaccines+and+gene+therapy+vectors.">The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.</a> Biotechnology Journal. 10 (5), 702-714 (2015).</li><li>Kumar, N., Pandey, D., Halder, A., Kumar, D., Gong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trends in Insect Molecular Biology and Biotechnology. , 163-191 (2018).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+fluorescent+probes+for+bioimaging+applications.">Development of fluorescent probes for bioimaging applications.</a> Proceedings of the Japan Academy. Series B. Physical and Biological Sciences. 86 (8), 837-847 (2010).</li><li>Hassan, M., Klaunberg, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+fluorescence+imaging+in+vivo.">Biomedical applications of fluorescence imaging in vivo.</a> Comparative Medicine. 54 (6), 635-644 (2004).</li><li>Yang, Z., Samanta, S., Yan, W., Yu, B., Qu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+for+biological+imaging.">Super-resolution microscopy for biological imaging.</a> Advances in Experimental Medicine and Biology. 3233, 23-43 (2021).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Methods in Cell Biology. 123, 77-94 (2014).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+fluorescence+imaging.">Live-cell fluorescence imaging.</a> Methods in Cell Biology. 114, 125-150 (2013).</li><li>Zheng, Q., Jockusch, S., Zhou, Z., Blanchard, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+reactive+oxygen+species+to+the+photobleaching+of+organic+fluorophores.">The contribution of reactive oxygen species to the photobleaching of organic fluorophores.</a> Photochemistry and Photobiology. 90 (2), 448-454 (2014).</li><li>Wojtovich, A. P., Foster, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+ROS+production.">Optogenetic control of ROS production.</a> Redox Biology. 2, 368-376 (2014).</li><li>Cai, D., Verhey, K. J., Meyhofer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+single+Kinesin+molecules+in+the+cytoplasm+of+mammalian+cells.">Tracking single Kinesin molecules in the cytoplasm of mammalian cells.</a> Biophysical Journal. 92 (12), 4137-4144 (2007).</li><li>Bohm, K. J., Stracke, R., Baum, M., Zieren, M., Unger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+temperature+on+kinesin-driven+microtubule+gliding+and+kinesin+ATPase+activity.">Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity.</a> FEBS Letters. 466, 59-62 (2000).</li><li>Porter, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+microtubule+movement+produced+by+sea+urchin+egg+kinesin.">Characterization of the microtubule movement produced by sea urchin egg kinesin.</a> The Journal of Biological Chemistry. 262 (6), 2794-2802 (1987).</li><li>Muyldermans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobodies:+natural+single-domain+antibodies.">Nanobodies: natural single-domain antibodies.</a> Annual Review of Biochemistry. 82, 775-797 (2013).</li><li>Verma, S., Kumar, N., Verma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+paclitaxel+on+critical+nucleation+concentration+of+tubulin+and+its+effects+thereof.">Role of paclitaxel on critical nucleation concentration of tubulin and its effects thereof.</a> Biochemical and Biophysical Research Communications. 478 (3), 1350-1354 (2016).</li><li>Parness, J., Horwitz, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxol+binds+to+polymerized+tubulin+in+vitro.">Taxol binds to polymerized tubulin in vitro.</a> Journal of Cell Biology. 91 (2), 479-487 (1981).</li><li>Schiff, P. B., Fant, J., Horwitz, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+microtubule+assembly+in+vitro+by+taxol.">Promotion of microtubule assembly in vitro by taxol.</a> Nature. 277 (5698), 665-667 (1979).</li><li>Kato, T., Kageshima, A., Suzuki, F., Park, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+human+(pro)renin+receptor+in+insect+cells+using+baculovirus+expression+system.">Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system.</a> Protein Expression and Purification. 58 (2), 242-248 (2008).</li><li>Liu, F., Wu, X., Li, L., Liu, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+baculovirus+expression+system+for+generation+of+virus-like+particles:+successes+and+challenges.">Use of baculovirus expression system for generation of virus-like particles: successes and challenges.</a> Protein Expression and Purification. 90 (2), 104-116 (2013).</li><li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+tag+protein+fusions:+from+molecular+and+biochemical+fundamentals+to+commercial+systems.">Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems.</a> Applied Microbiology and Biotechnology. 60 (5), 523-533 (2003).</li><li>Huang, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+paclitaxel+induces+fewer+hematopoietic+and+cardiovascular+complications+than+bioequivalent+doses+of+Taxol.">Liposomal paclitaxel induces fewer hematopoietic and cardiovascular complications than bioequivalent doses of Taxol.</a> International Journal of Oncology. 53 (3), 1105-1117 (2018).</li></ol>

The authors have no competing financial interests to declare.

The Sf9-baculovirus expression system is one of the most versatile and successful methods for high-throughput protein production19,36,37. The posttranslational modification ability of Sf9 cells is highly comparable to the mammalian system15. A considerable disadvantage of using this system is that it is slow and sensitive to contamination. One of the most critical steps is efficient infection and successf...

An immensely crowded cell environment poses many challenges in sorting destined proteins and molecules. This intense workload of organization and spatiotemporal distribution of molecules within the cytoplasm is facilitated by molecular motors and cytoskeletal tracks. Molecular motors are the enzymes that hydrolyze the energy currencies such as ATP and utilize that energy during motion and force generation1. Based on the amino acid sequence similarity, kinesins are grouped into 14 families and despite this similarity, each motor contributes uniquely to the functioning of a cell. Kinesin-3 family motors constitute one of the largest, comprising five subfamilies (KIF1, KIF13, KIF14, KIF16, and KIF28)2, associated with diverse cellular and physiological functions, including vesicle transport, signaling, mitosis, nuclear migration, and development 3,4,5. Impairment in kinesin-3 transport function implicates in many neurodegenerative disorders, developmental defects, and cancer diseases6,7,8,9.
Recent work has demonstrated that kinesin-3 motors are monomers but undergo cargo-induced dimerization and result in fast and superprocessive motility compared to conventional kinesin10,11,12,13. Their biochemical and biophysical characterization needs a large quantity of purified, active proteins. However, their production in the prokaryotic expression system resulted in inactive or aggregated motors, presumably due to incompatible protein synthesis, folding and modification machinery14,15,16,17,18. To circumvent such limitations and increase the yield, here we have established a robust Sf9-baculovirus expression system to express and purify these motors.
The baculovirus expression system uses Sf9 insect cell lines as a host system for high-throughput eukaryotic recombinant protein expression19,20. Baculovirus possesses a strong polyhedrin promoter that assists in heterologous gene expression and the production of soluble recombinant proteins17. Due to its cost-effectiveness, safe to handle and high amount of active protein expression, it has become a powerful tool21. To express a protein of interest, a key step is to generate a recombinant bacmid. Since the commercially available bacmid generating kits are expensive and we will be working with more samples, we developed an in-house protocol for both large and small inserts of kinesin-3 motors into bacmids. Sf9-purified kinesin-3 motors were used to characterize in vitro single-molecule and multi-motor microtubule gliding properties using total internal reflection fluorescence (TIRF) microscopy. Motors are C-terminally tagged with 3-tandem fluorescent molecules (3xmCit) to provide enhanced signal and decreased photobleaching. Due to its increased signal-to-noise ratio, less phototoxicity, and selective imaging of a very small area close to the coverslip, TIRF imaging has been widely used to visualize protein dynamics at the single-molecule level in vivo and in vitro.
This study discusses the purification of kinesin-3 motors by employing Sf9-baculovirus expression system and in vitro single-molecule imaging and multi-motor gliding analysis of motors using TIRF microscopy. Altogether, this study shows that the motility properties of Sf9 purified motors are identical to that of motors prepared from mammalian cell lysates. Hence, we believe that the Sf9-baculovirus system can be adapted to express and purify any motor protein of interest.

<table><tbody><tr><td>&lt;strong&gt;Sf9 culture and transfection materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-FLAG M2 affinity</td><td>Biolegend</td><td>651502</td><td>For protein purification</td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A6279</td><td>For protein purification</td></tr><tr><td>Cellfectin</td><td>Invitrogen</td><td>10362100</td><td>For Sf9 transfection</td></tr><tr><td>DTT</td><td>Sigma</td><td>D5545</td><td>For motility assays and protein purification</td></tr><tr><td>FLAG peptide</td><td>Sigma</td><td>F3290</td><td>For protein purification</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>To freeze the protein</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>For Sf9 lysis buffer</td></tr><tr><td>IGEPAL CA 630</td><td>Sigma</td><td>I8896</td><td>For Sf9 lysis buffer</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td>For buffers preparation</td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td>For protein purification</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2670</td><td>For buffers preparation</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td>For preparing lysis buffer</td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td>For protein purification</td></tr><tr><td>Sf9 cells</td><td>Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India).</td><td></td><td>For baculovirs expression and protein purification</td></tr><tr><td>Sf9 culture bottles</td><td>Thermo Scientific</td><td>4115-0125</td><td>For suspension culture</td></tr><tr><td>Sf-900/SFM medium (1X)</td><td>Thermo Scientific</td><td>10902-096 -500ml</td><td>For culturing Sf9 cells</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S1888</td><td>Preparing lysing buffer for Sf9 cells</td></tr><tr><td>Unsupplemented Grace&amp;rsquo;s media</td><td>Thermo Scientific</td><td>11595030 -500ml</td><td>For Sf9 transfection</td></tr><tr><td>&lt;strong&gt;Mirotubule Polymerization and Single molecule assay materails&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A2647</td><td>For motility and gliding assay</td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td>For blocking motility chamber</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C9322</td><td>For motility and gliding assay</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D5879</td><td>For dissolving Rhodamine</td></tr><tr><td>EGTA</td><td>Sigma</td><td>3777</td><td>For preparing buffers</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td>For motility and gliding assay</td></tr><tr><td>Glucose oxidase</td><td>Sigma</td><td>G2133</td><td>For motility and gliding assay</td></tr><tr><td>GTP</td><td>Sigma</td><td>G8877</td><td>For microtubule polymerization</td></tr><tr><td>KOH</td><td>Sigma</td><td>P1767</td><td>Preparing PIPES buffer pH 6.9</td></tr><tr><td>PIPES</td><td>Sigma</td><td>P6757</td><td>For preparing motility and gliding assay buffers</td></tr><tr><td>&lt;strong&gt;Microtubule gliding assay materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>26G&amp;nbsp; needle</td><td>Dispovan</td><td></td><td>For shearing microtubules</td></tr><tr><td>Casein</td><td>Sigma</td><td>C3400</td><td>For microtubule glidning assay</td></tr><tr><td>GFP nanobodies</td><td>Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA)</td><td></td><td>For attaching motors to the coverslip</td></tr><tr><td>Rhodamine</td><td>Thermo Scientific</td><td>46406</td><td>For preparing labelling tubulin</td></tr><tr><td>&lt;strong&gt;Microscope and other instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.5ml, 1.5 and 2-ml microcentrifuge tubes</td><td>Eppendorf</td><td></td><td>For Sf9 culture and purification</td></tr><tr><td>10ml&amp;nbsp; disposable sterile pipettes</td><td>Eppendorf</td><td></td><td>For Sf9 culture and purification</td></tr><tr><td>10ul, 200ul, 1ml micropipette tips</td><td>Eppendorf</td><td></td><td>For Sf9 culture and purification</td></tr><tr><td>15ml concal tubes</td><td>Eppendorf</td><td></td><td>For Sf9 culture and purification</td></tr><tr><td>35mm cell culture dish</td><td>Cole Palmer</td><td>15179-39</td><td>For Sf9 culture</td></tr><tr><td>Balance</td><td>Sartorious</td><td></td><td>0.01g-300g</td></tr><tr><td>Benchtop orbial shaking incubator</td><td>REMI</td><td></td><td>For Sf9 suspenculture at 28oC</td></tr><tr><td>Camera</td><td>EMCCD Andor iXon Ultra 897</td><td></td><td>For TIRF imaging and acquesition</td></tr><tr><td>Double sided tape</td><td>Scotch</td><td></td><td>For making motility chamber</td></tr><tr><td>Glass coverslip</td><td>Fisherfinest</td><td>12-548-5A</td><td>size; 22X30</td></tr><tr><td>Glass slide</td><td>Blue Star</td><td></td><td>For making motility chamber</td></tr><tr><td>Heating block</td><td>Neuation</td><td></td><td>Dissolving paraffin wax</td></tr><tr><td>Inverted microscope</td><td>Nikon Eclipse Ti- U</td><td></td><td>To check protein expression</td></tr><tr><td>Lasers</td><td>488nm (100mW)</td><td></td><td>For TIRF imaging</td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>For sample freezing and storage</td></tr><tr><td>Microcapillary loading tip</td><td>Eppendorf</td><td>EP022491920</td><td>For shearing microtubules</td></tr><tr><td>Microscope</td><td>Nikon Eclipse Ti2-E with DIC set up</td><td></td><td>For TIRF imaging</td></tr><tr><td>Mini spin</td><td>Genetix, BiotechAsia Pvt.Ltd</td><td></td><td>For quick spin</td></tr><tr><td>Objective</td><td>100X TIRF objective with 1.49NA oil immersion</td><td></td><td>For TIRF imaging</td></tr><tr><td>Optima UltraCentrifuge XE</td><td>Beckman Coulter</td><td></td><td>For protein purification</td></tr><tr><td>Parafilm</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>pH-meter</td><td>Corning</td><td>Coring 430</td><td>To adjust pH</td></tr><tr><td>Pipette-boy</td><td>VWR</td><td></td><td>For Sf9 culture and purification</td></tr><tr><td>Sorvall Legend Micro 21</td><td>Thermo Scientific</td><td></td><td>For protein purification</td></tr><tr><td>Sorvall ST8R centrifuge</td><td>Thermo Scientific</td><td></td><td>Protein purification</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td></td><td>For microtubule polymerization</td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman coulter</td><td></td><td>SW60Ti rotor</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman</td><td></td><td>5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm</td></tr><tr><td>Vortex mixer</td><td>Neuation</td><td></td><td>Sample mixing</td></tr><tr><td>Wax</td><td>Sigma</td><td>V001228</td><td>To seal motility chamber</td></tr></tbody></table>

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.

To express and purify active and functional recombinant motor proteins at a large scale using the Sf9-baculovirus expression, the system needs generation of viral particles stably carrying a coding sequence to infect Sf9 cells. To achieve this, Sf9 cells were transfected with recombinant bacmid encoding KIF1A(1-393LZ)-3xmCit-FLAG. After 72 h, a significant population of cells showed expression of green fluorescent protein (mCitrine) with enlarged cells and nuclei (Figure 1 and <strong class=...

Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

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

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 ...

single-molecule-analysis-sf9-purified-superprocessive-kinesin-3

Research

JoVE Journal

Biochemistry

1.9K Views.  Indian Institute of Technology Gandhinagar. 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.

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

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 ...

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.

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.

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

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Neuroscience

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

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 ...

Biology

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

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 ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

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

Summary

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Chapters in this video

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

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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

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

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Isolation and Purification of Kinesin from Drosophila Embryos

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy