Name: motility of single molecules and clusters of bi-directional kinesin-5 cin8 purified from s. cerevisiae cells
Uploaded: 2022-02-02T14:00:00
Description: Watch this Scientific Journal Video about Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells at JoVE.com

This research was supported in part by the Israel Science Foundation grant (ISF-386/18) and the Israel Binational Science Foundation grant (BSF-2019008), awarded to L.G.

1. Preparation of buffers and reagents
<ol>
	<li>Buffers
	<ol>
		<li>-Leu aa dropout mix: Mix 2 g each of Adenine, Uracil, Tryptophan, Histidine, Lysine, and Methionine and store at room temperature.</li>
		<li>Yeast selective medium with raffinose (1 L): Mix 6.7 g of yeast nitrogen base (with ammonium sulfate), 2 g of -Leu aa dropout mix, and 20 g of raffinose in double-distilled water by stirring (without heating) until fully dissolved. Using a 0.22 &#181;m filter, filter the solution into a sterile ...

<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=Two+Saccharomyces+cerevisiae+kinesin-related+gene+products+required+for+mitotic+spindle+assembly.">Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.</a> Journal of Cell Biology. 118 (1), 109-120 (1992).</li><li>Gerson-Gurwitz, A., 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=Mid-anaphase+arrest+in+S.+cerevisiae+cells+eliminated+for+the+function+of+Cin8+and+dynein.">Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein.</a> Cellular and Molecular Life Sciences. 66 (2), 301-313 (2009).</li><li>Fridman, V., Gerson-Gurwitz, A., Movshovich, N., Kupiec, M., Gheber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midzone+organization+restricts+interpolar+microtubule+plus-end+dynamics+during+spindle+elongation.">Midzone organization restricts interpolar microtubule plus-end dynamics during spindle elongation.</a> EMBO Reports. 10 (4), 387-393 (2009).</li><li>Movshovich, N., 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=Slk19-dependent+mid-anaphase+pause+in+kinesin-5-mutated+cells.">Slk19-dependent mid-anaphase pause in kinesin-5-mutated cells.</a> Journal of Cell Science. 121 (15), 2529-2539 (2008).</li><li>Gerson-Gurwitz, A., 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=Directionality+of+individual+kinesin-5+Cin8+motors+is+modulated+by+loop+8,+ionic+strength+and+microtubule+geometry.">Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry.</a> Embo Journal. 30 (24), 4942-4954 (2011).</li><li>Roostalu, J., 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=Directional+switching+of+the+kinesin+Cin8+through+motor+coupling.">Directional switching of the kinesin Cin8 through motor coupling.</a> Science. 332 (6025), 94-99 (2011).</li><li>Shapira, O., Goldstein, A., Al-Bassam, J., Gheber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+physiological+role+for+bi-directional+motility+and+motor+clustering+of+mitotic+kinesin-5+Cin8+in+yeast+mitosis.">A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis.</a> Journal of Cell Science. 130 (4), 725-734 (2017).</li><li>Goldstein-Levitin, A., Pandey, H., Allhuzaeel, K., Kass, I., Gheber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+functions+and+motile+properties+of+bi-directional+kinesin-5+Cin8+are+regulated+by+neck+linker+docking.">Intracellular functions and motile properties of bi-directional kinesin-5 Cin8 are regulated by neck linker docking.</a> eLife. 10, 71036 (2021).</li><li>Pandey, H., 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=Drag-induced+directionality+switching+of+kinesin-5+Cin8+revealed+by+cluster-motility+analysis.">Drag-induced directionality switching of kinesin-5 Cin8 revealed by cluster-motility analysis.</a> Science Advances. 7 (6), 1687 (2021).</li><li>Pandey, H., Popov, M., Goldstein-Levitin, A., Gheber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+by+which+kinesin-5+motors+perform+their+multiple+intracellular+functions.">Mechanisms by which kinesin-5 motors perform their multiple intracellular functions.</a> International Journal of Molecular Sciences. 22 (12), 6420 (2021).</li><li>Fridman, 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-5+Kip1+is+a+bi-directional+motor+that+stabilizes+microtubules+and+tracks+their+plus-ends+in+vivo.">Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo.</a> Journal of Cell Science. 126, 4147-4159 (2013).</li><li>Edamatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+motility+of+the+fission+yeast+kinesin-5,+Cut7.">Bidirectional motility of the fission yeast kinesin-5, Cut7.</a> Biochemical and Biophysical Research Communications. 446 (1), 231-234 (2014).</li><li>Popchock, A. R., 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=The+mitotic+kinesin-14+KlpA+contains+a+context-dependent+directionality+switch.">The mitotic kinesin-14 KlpA contains a context-dependent directionality switch.</a> Nature Communications. 8, 13999 (2017).</li><li>Bodrug, 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=The+kinesin-5+tail+domain+directly+modulates+the+mechanochemical+cycle+of+the+motor+domain+for+anti-parallel+microtubule+sliding.">The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.</a> eLife. 9 (9), 51131 (2020).</li><li>Gheber, L., Kuo, S. C., Hoyt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+properties+of+the+kinesin-related+Cin8p+spindle+motor+extracted+from+Saccharomyces+cerevisiae+cells.">Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells.</a> Journal of Biological Chemistry. 274 (14), 9564-9572 (1999).</li><li>Pandey, H., 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=Flexible+microtubule+anchoring+modulates+the+bi-directional+motility+of+the+kinesin-5+Cin8.">Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.</a> Cellular and Molecular Life Sciences. 78 (16), 6051-6068 (2021).</li><li>Shapira, O., Gheber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+properties+of+the+bi-directional+kinesin-5+Cin8+are+affected+by+phosphorylation+in+its+motor+domain.">Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain.</a> Scientific Reports. 6, 25597 (2016).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Britto, 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=Schizosaccharomyces+pombe+kinesin-5+switches+direction+using+a+steric+blocking+mechanism.">Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (47), 7483-7489 (2016).</li><li>Kapitein, L. C., 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=Microtubule+cross-linking+triggers+the+directional+motility+of+kinesin-5.">Microtubule cross-linking triggers the directional motility of kinesin-5.</a> Journal of Cell Biology. 182 (3), 421-428 (2008).</li><li>Furuta, K., Edamatsu, M., Maeda, Y., Toyoshima, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+directed+movement+in+vitro+motile+properties+of+fission+yeast+kinesin-14+Pkl1.">Diffusion and directed movement in vitro motile properties of fission yeast kinesin-14 Pkl1.</a> Journal of Biological Chemistry. 283 (52), 36465-36473 (2008).</li><li>Katrukha, E. A., 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=Probing+cytoskeletal+modulation+of+passive+and+active+intracellular+dynamics+using+nanobody-functionalized+quantum+dots.">Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots.</a> Nature Communications. 8, 14772 (2017).</li><li>Tinevez, J. Y., 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=TrackMate:+An+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: An open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2017).</li><li>Jakobs, M. A. 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In this work, a protocol for single-molecule motility assay with the bi-directional kinesin-5 Cin8 and the motility analysis are presented. The full-length Cin818 including the native nuclear localization signal (NLS) at the C-terminal has been purified from the native host S. cerevisiae. As the Cin8 is a nuclear motor protein, grinding the S. cerevisiae cells under liquid nitrogen is found to be the most efficient method for cell lysis. After lysis, by combining metal affinity a...

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

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

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

A large number of motility events within eukaryotic cells are mediated by the function of molecular motor proteins. These motors move along the cytoskeletal filaments, actin filaments, and microtubules (MTs), and convert the chemical energy of ATP hydrolysis into kinetic and mechanical forces required to drive biological motility within cells. The MT-based S. cerevisiae Cin8 is a bipolar, homotetrameric kinesin-5 motor protein that crosslinks and slides spindle MTs apart1. Cin8 performs essential functions during mitosis, in spindle assembly2,3,4 and spindle elongation during anaphase5,6,7. Previously, it had been demonstrated that Cin8 is a bi-directional motor, which switches directionality under different experimental conditions. For instance, under high ionic strength conditions, single Cin8 motors move toward the minus-end of the MTs, while in clusters, in multi-motor MT gliding assays, and between antiparallel MTs, Cin8 motors move mainly toward the plus-ends of the MTs8,9,10,11,12. These findings were highly unexpected because of several reasons. First, Cin8 carries its catalytic motor domain at the amino-terminus and such motors were previously believed to be exclusively plus-end directed, whereas Cin8 was shown to be minus-end directed at the single-molecule level. Second, kinesin motors were believed to be unidirectional, either minus-end or plus-end directed, whereas Cin8 was shown to be bi-directional, depending on the experimental conditions. Finally, because of the MT orientation at the mitotic spindle, the classical role of kinesin-5 motors in the separation of spindle poles during spindle assembly and anaphase B could only be explained by their plus-end directed motility on the MTs they crosslink1,13. Following the first reports on the bi-directionality of Cin8, a few other kinesin motors were demonstrated to be bi-directional14,15,16, indicating that the bi-directional motility of kinesin motors may be more common than earlier believed.
It has been previously reported that in cells, Cin8 also moves in a bi-directional manner8, supporting the notion that the bi-directional motility of some kinesin-5 motors is important for their intracellular functions. In addition, since the three kinesin-5 motors that were reported to be bi-directional are from fungal cells, a possible role for the bi-directionality of kinesin-5 motors has been recently proposed in such cells10. According to this model, in closed mitosis of fungal cells, where the nuclear envelope doesn&#39;t break down during mitosis, kinesin-5 motors provide the initial force that separates the spindle poles apart prior to spindle assembly. To perform this task, prior to spindle pole separation, kinesin-5 motors localize near the spindle poles, by their minus-end directed motility on single nuclear MTs. Once at this position, kinesin-5 motors cluster, switch directionality, capture, and cross-link MTs from neighboring spindle poles. Subsequently, kinesin-5 motors provide the initial separation of the poles by plus-end directed motility on the MTs they crosslink. By this model, both minus-end directed motility on single MTs and plus-end directed motility on cross-linked MTs during antiparallel sliding are required for fungal kinesin-5 motors to perform their roles in spindle assembly1,13.
The overall goal of the described method is to obtain high-purity fungal GFP-tagged kinesin-5 Cin8 and to perform single-molecule motility assays (Figure 1) while separately analyzing the motility of single molecules and clusters of Cin8. The separation between single molecules and clusters is important since one of the factors that had been demonstrated to affect the directionality of Cin8 is its accumulation in clusters on the MTs10,12. Alternative motility assays, such as the MT surface gliding and MT sliding assays do not provide information regarding the activity of single motor proteins17,18. The robust single-molecule motility assay and analysis methods described here have been successfully applied to characterize different aspects of kinesin-5 motors, Cin8 and Kip110,11,12,14,19,20.
Here, a detailed protocol is presented for Cin8 overexpression and purification, polymerization of MTs, and the single-molecule motility assay. Furthermore, the analyses to differentiate between single molecules and clusters of Cin8, and to determine single motor and cluster velocities by mean displacement (MD) and mean square displacement (MSD) analysis are also described. This protocol aims to help researchers to visualize all the steps of the procedures and assist with troubleshooting this type of assays.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63425/63425fig01v2.jpg" /> 
Figure 1: Schematic representation of the single-molecule motility assay. Biotinylated fluorescent MTs are attached to the glass surface, coated with Avidin that interacts with the surface-attached biotinylated-BSA. The green arrow represents the movement direction of single Cin8 molecules under high ionic strength conditions. +/- represent the polarity of the MT. <a href="https://www.jove.com/files/ftp_upload/63425/63425fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenine</td>
			<td>FORMEDIUM</td>
			<td>DOC0230</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-BSA</td>
			<td>Sigma</td>
			<td>A8549</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C7078</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase (C40)</td>
			<td>Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine-Kinase</td>
			<td>Sigma</td>
			<td>C3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter set for GFP</td>
			<td>Chroma</td>
			<td>49002: ET-EGFP (FITC/Cy2)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter set for Rhodamine</td>
			<td>Chroma</td>
			<td>49004: ET-CY3/TRITC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence inverted microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200M</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Tivan Biotech</td>
			<td>GAL02</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Sigma</td>
			<td>G7141</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>GlycylGlycine</td>
			<td>Merck</td>
			<td>G0674</td>
			<td></td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jana Bioscience</td>
			<td>Nu-405L</td>
			<td></td>
		</tr>
		<tr>
			<td>GTB</td>
			<td>Cytoskeleton</td>
			<td>BST01-010</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>Duchefa Biochemie</td>
			<td>H0710.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ-FIJI software</td>
			<td>https://imagej.net/plugins/trackmate/</td>
			<td>version 2.1.0/1.53c; Java 1.8.0_172 [64-bit] for Windows 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma</td>
			<td>I0125</td>
			<td></td>
		</tr>
		<tr>
			<td>InstantBlue Coomassie Protein Stain</td>
			<td>Abcam</td>
			<td>ab119211</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Zeiss</td>
			<td>100x/1.4 oil DIC objective</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>FORMEDIUM</td>
			<td>DOC0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>Duchefa Biochemie</td>
			<td>M0715.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neo</td>
			<td>Andor Technologies</td>
			<td>sCMOS camera</td>
			<td></td>
		</tr>
		<tr>
			<td>NeutraAvidin</td>
			<td>Life</td>
			<td>A2666</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Invitrogen</td>
			<td>R901-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Creatine</td>
			<td>Sigma</td>
			<td>P1937</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid F-127 (poloxamer)</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Tivan Biotech</td>
			<td>RAF01</td>
			<td></td>
		</tr>
		<tr>
			<td>Size Exclusion chromatography instument</td>
			<td>GE Healthcare</td>
			<td>AKTA Pure</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>NanoDrop</td>
			<td></td>
		</tr>
		<tr>
			<td>Superose-6 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Roshe</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Duchefa Biochemie</td>
			<td>T0720.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin protein</td>
			<td>Cytoskeleton</td>
			<td>T240</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin, biotinylated</td>
			<td>Cytoskeleton</td>
			<td>T333P</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin, TRITC Rhodamine</td>
			<td>Cytoskeleton</td>
			<td>TL530M</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base</td>
			<td>FORMEDIUM</td>
			<td>CYN0401S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-GFP antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC8036</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

The experiment aims to investigate the motility characteristics of bi-directional motor protein Cin8 of different cluster sizes on single MTs. Representative motility of Cin8-GFP is also evident from the kymographs in Figure 5A, where the spatial position of the motor over time is shown.
For the analysis of the motile properties of Cin8-GFP, first, the cluster size is assigned (step 4.3) to each MT-attached motile Cin8-GFP particle, and then the position of the ex...

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

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

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

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

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

The Saccharomyces cerevisiae kinesin-5 Cin8 is a bidirectional motor protein. Cin8 moves in the minus-end direction of the microtubules as a single molecule, and changes directionality under a number of different experimental conditions. The overall goal of our research is to understand the mechanism of bidirectional motility by studying factors and structural elements that regulate the bidirectional motility of Cin8.This protocol comprehensively explains the purification of GFP-type full-length Cin8 overexpressed in yeast cells, the single-molecule motility assay, and the subsequent analysis of motile properties of single molecules and clusters of Cin8. This separation is important, since the directionality of Cin8 is affected by its accumulation in clusters on the microtubule. This technique can be easily adapted to purify other nuclear proteins from the yeast.Additionally, it can be applied to any fluorescent particles that need to be separated into size groups based on their fluorescence intensity. Demonstrating the procedure will be Dr.Mary Popov, our lab manager, Dr.Alina Goldstein-Levitin, and Dr.Nurit Siegler, postdoctoral fellows;Tatiana Zvagelsky, Mayan Sadan, and Shira Hershfinkel, graduate students;and Neta Yanir, Yahel Abraham, Roy Avraham, and Meital Haberman, undergraduate students from our laboratory. To begin, grow Saccharomyces cerevisiae cells containing the plasmid for overexpression of Cin8-GFP-6His to the exponential growth phase in one liter of yeast-selective medium, supplemented with 2%raffinose at 28 degrees Celsius.Then induce Cin8-GFP-6His overexpression by adding 2%galactose, and monitor the yeast culture growth by measuring absorbance at 600 nanometers. Five hours after galactose addition, harvest the cells by centrifugation. Re-suspend the cells in lysis buffer, and freeze them in liquid nitrogen.Next, using a chilled mortar and pestle, grind the frozen cells in liquid nitrogen. Keep adding liquid nitrogen during the grinding, so that the extracts remain frozen. Monitor the cell lysis under a phase or differential interference contrast microscope.Then thaw the ground cells, and centrifuge them. Load the supernatant onto a gravity-flow column filled with two milliliters of Nickel-NTA, and pre-equilibrate it with lysis buffer. Let the supernatant flow out through the column.Wash the column with five column volumes of lysis buffer, followed by five column volumes of lysis buffer supplemented with 25-millimolar imidazole. Then elute the Cin8-GFP-6His with elution buffer. Analyze the eluted samples by SDS-PAGE fractionation, followed by Coomassie blue staining and Western blot analysis probed with anti-GFP antibody.After pooling the fractions containing Cin8-GFP-6His, purify them by size-exclusion chromatography at a flow rate of 0.5 milliliters per minute, and a column pressure limit of 1.5 megapascals. Simultaneously monitor the absorbance at 280 nanometers. Collect the fractions corresponding to the Cin8-GFP tetramer and analyze them by SDS-PAGE and Western blotting.Then aliquot the selected fractions, snap-freeze, and store until use at minus 80 degrees Celsius. To polymerize and stabilize the biotin and rhodamine labeled microtubules, mix the reaction components in a 1.5-milliliter tube, and incubate the mixture for one hour at 37 degrees Celsius. Then add 80 microliters of warm general tubulin buffer, mix carefully, and centrifuge.Discard the supernatant, and re-suspend the pellet carefully by pipetting up and down with 50 microliters of warm general tubulin buffer. Then store the suspension at 28 or 37 degrees Celsius. Examine the microtubules with a fluorescence microscope, using the 647-nanometer rhodamine channel.Next, remove the protective paper from the tape strips and place a silanized coverslip on the strips to create three 10-microliter volume flow chambers. Then perform the avidin coating of the coverslip by sequential addition of the indicated reagents. Incubate the coverslip for three to five minutes before washing it with 80 microliters of general tubulin buffer.Next, attach the biotinylated microtubules to the b-BSA/avidin-coated coverslips by inserting 20 microliters of microtubules into the flow chamber. Incubate the slides with the coverslip facing downwards in a dark humidity chamber for five minutes at room temperature. Then wash the slides with 200 microliters of general tubulin buffer.Next, apply 30 microliters of motility reaction mix followed by Cin8-GFP motors diluted in 20 microliters of the motility reaction mix into the flow chamber, and immediately proceed to image the movement of the motors along the microtubules. For motor motility imaging, place a drop of immersion oil on the microscope objective, then place the flow chamber on the fluorescent microscope stage, with the coverslip facing the objective. Turn on the rhodamine channel to focus on the microtubules attached to the coverslip surface.Then, using ImageJ Micro-Manager software, acquire the image with a 20-millisecond exposure. Next, turn on the GFP channel and acquire 90 time-lapse images with a one-second interval and 800-millisecond exposure. In the ImageJ Fiji software, open the time-lapse movie and the corresponding microtubule field image.Synchronize these two windows by choosing Analyze, followed by Tools and Synchronize Windows. To obtain a kymograph, highlight one microtubule using the Segmented Line option. Then, from Analyze, select the Multi Kymograph tab.To determine the cluster size of the Cin8-GFP molecules, perform the background subtraction and correction for uneven illumination by clicking on Process, followed by Subtract Background. Set the Rolling ball radius to 100 pixels, and check the Sliding paraboloid option. Then track a specific non-motile Cin8-GFP motor using the TrackMate plugin of the ImageJ Fiji software.Choose Plugins, followed by Tracking, TrackMate, LoG detector, and Simple LAP tracker to follow the mean fluorescence intensity of the Cin8-GFP motor as a function of time within a circle of four pixels radius. After repeating the procedure for different Cin8-GFP motors, plot the fluorescence intensities as a function of time. Next, for tracking the motility of the Cin8-GFP molecules along the microtubule tracks, crop the microtubules to be analyzed in the time-lapse sequence of recorded frames by highlighting it with the rectangle tool and clicking Image, followed by Crop.Choose a fluorescent Cin8-GFP particle for the analysis. Record the particle coordinates in each frame of the time-lapse sequence using the point tool and Measure options. Similarly, record coordinates for other fluorescent particles in the time-lapse sequence.Then assign cluster size to all the examined Cin8-GFP particles in the first frame of their appearance, as demonstrated earlier. Next, using the indicated formula and the determined Cin8-GFP movement coordinates, calculate the displacements of Cin8-GFP at each time point with respect to the initial coordinates. From these displacement values, calculate the displacement for all possible time intervals for a specified Cin8-GFP particle.Repeat the procedure for all the examined Cin8-GFP particles. Finally, plot the mean displacement of all examined Cin8-GFP particles versus time interval, and subject it to a linear fit. The slope of this fit represents the mean velocity of motile Cin8-GFP particles.The motility characteristics of bidirectional motor protein Cin8 of different cluster sizes on single microtubules are shown here. For each cluster size category, more than 40 trajectories of individual Cin8-GFP were extracted from the recordings. Plotting the mean displacements of single molecules and clusters of Cin8 motors as a function of the time interval demonstrated that single Cin8-GFP molecules move in a unidirectional minus-end-directed manner with high velocity.In contrast, Cin8 clusters exhibit lower velocity and higher bidirectional motility. While performing this procedure, high-purity motor protein must be used to avoid any contaminations that can affect motor clustering. In analyzing the motor fluorescence intensity, it is important to examine the motors in the first frame they appear, to avoid the effect of photobleaching.When studying the motility of bidirectional motors, it is extremely important to separately analyze single molecules and clusters, as motor clustering is one of the key factors that affect motor directionality.

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