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>

<ol>
	<li>Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+motor+toolbox+for+intracellular+transport.">The molecular motor toolbox for intracellular transport.</a> Cell. 112, 467-480 (2003).</li><li>Miki, H., Okada, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+kinesin+superfamily:+insights+into+structure+and+function.">Analysis of the kinesin superfamily: insights into structure and function.</a> Trends in Cell Biology. 15 (9), 467-476 (2005).</li><li>Hirokawa, N., Niwa, S., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+in+neurons:+transport+mechanisms+and+roles+in+brain+function,+development,+and+disease.">Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.</a> Neuron. 68 (4), 610-638 (2010).</li><li>Hirokawa, N., Takemura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+and+mechanisms+of+directional+transport+in+neurons.">Molecular motors and mechanisms of directional transport in neurons.</a> Nature Reviews Neuroscience. 6 (3), 201-214 (2005).</li><li>Patel, N. M., 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=KIF13A+motors+are+regulated+by+Rab22A+to+function+as+weak+dimers+inside+the+cell.">KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.</a> Scientific Advances. 7 (6), (2021).</li><li>Franker, M. A., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-based+transport+-+basic+mechanisms,+traffic+rules+and+role+in+neurological+pathogenesis.">Microtubule-based transport - basic mechanisms, traffic rules and role in neurological pathogenesis.</a> Journal of Cellular Science. 126, 2319-2329 (2013).</li><li>Gunawardena, S., Anderson, E. N., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+and+neurodegenerative+disease:+vesicle-motor+complex+formation+and+their+regulation.">Axonal transport and neurodegenerative disease: vesicle-motor complex formation and their regulation.</a> Degernative Neurological and Neuromuscular Disease. 4, 29-47 (2014).</li><li>Rath, O., Kozielski, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesins+and+cancer.">Kinesins and cancer.</a> Nature Reviews Cancer. 12 (8), 527-539 (2012).</li><li>Wang, Z. Z., 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=KIF14+promotes+cell+proliferation+via+activation+of+Akt+and+is+directly+targeted+by+miR-200c+in+colorectal+cancer.">KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer.</a> International Journal of Oncology. 53 (5), 1939-1952 (2018).</li><li>Guo, S. K., Shi, X. X., Wang, P. Y., Xie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Run+length+distribution+of+dimerized+kinesin-3+molecular+motors:+comparison+with+dimeric+kinesin-1.">Run length distribution of dimerized kinesin-3 molecular motors: comparison with dimeric kinesin-1.</a> Scientific Reports. 9 (1), 16973 (2019).</li><li>Scarabelli, G., 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=Mapping+the+processivity+determinants+of+the+Kinesin-3+motor+domain.">Mapping the processivity determinants of the Kinesin-3 motor domain.</a> Biophysical Journal. 109 (8), 1537-1540 (2015).</li><li>Soppina, V., 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=Dimerization+of+mammalian+kinesin-3+motors+results+in+superprocessive+motion.">Dimerization of mammalian kinesin-3 motors results in superprocessive motion.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (15), 5562-5567 (2014).</li><li>Soppina, V., Verhey, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+family-specific+K-loop+influences+the+microtubule+on-rate+but+not+the+superprocessivity+of+kinesin-3+motors.">The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors.</a> Molecular Biology Cell. 25 (14), 2161-2170 (2014).</li><li>Soppina, V., 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=Kinesin-3+motors+are+fine-tuned+at+the+molecular+level+to+endow+distinct+mechanical+outputs.">Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs.</a> BMC Biology. , (2022).</li><li>Korten, T., Chaudhuri, S., Tavkin, E., Braun, M., Diez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-1+Expressed+in+insect+cells+improves+microtubule+in+vitro+gliding+performance,+long-term+stability+and+guiding+efficiency+in+nanostructures.">Kinesin-1 Expressed in insect cells improves microtubule in vitro gliding performance, long-term stability and guiding efficiency in nanostructures.</a> IEEE Transactions on Nanobioscience. 15 (1), 62-69 (2016).</li><li>Schmidt, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+expression+systems+in+the+pharmaceutical+industry.">Recombinant expression systems in the pharmaceutical industry.</a> Applied Microbiology and Biotechnology. 65 (4), 363-372 (2004).</li><li>Kurland, C., Gallant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Errors+of+heterologous+protein+expression.">Errors of heterologous protein expression.</a> Current Opinions in Biotechnology. 7 (5), 489-493 (1996).</li><li>Tao, L., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+assay+of+mitotic+motors.">Purification and assay of mitotic motors.</a> Methods. 51 (2), 233-241 (2010).</li><li>Kost, T. A., Condreay, J. P., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nature Biotechnology. 23 (5), 567-575 (2005).</li><li>Felberbaum, R. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sf9 culture and transfection materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG M2 affinity</td>
			<td>Biolegend</td>
			<td>651502</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>For motility assay and 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 assay mixture</td>
		</tr>
		<tr>
			<td>FLAG peptide</td>
			<td>Sigma</td>
			<td>F3290</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>Preparing lysing Sf9 cells</td>
		</tr>
		<tr>
			<td>IGEPAL CA 630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Preparing lysing buffer for Sf9 cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>For motility assay and purification</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>For preparing lysis buffer</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 motility 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 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&#8217;s media</td>
			<td>Thermo Scientific</td>
			<td>11595030 -500ml</td>
			<td>For Sf9 transfection</td>
		</tr>
		<tr>
			<td>Mirotubule Polymerization and Single molecule assay materails</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 motility and gliding assay</td>
		</tr>
		<tr>
			<td>Microtubule gliding assay materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#160; 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>Microscope and other instruments</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&#160; 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

1.8K 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

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.