Name: single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors
Uploaded: 2022-07-27T13:00:00
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>
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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. 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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 ...

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