Roche's Publication Grant covered the Free Access publication and production of this article. The author also thanks E. Premkumar Reddy, Richard V. Mettus, Stephen C. Cosenza, Sau Ying Yip and Sol D. Gloria for their support and critical reading of the manuscript.

1. Cloning of the tdTomato-tagged Wild-type and Motorless KIF5B Proteins
<ol>
	<li>Amplify the cDNAs for the human wild-type and motorless KIF5B proteins using the primers in Table 1, Taq DNA polymerase (5 units for 100 &#956;l), dNTP mix (2 mM for each deoxynucleotide) and its 10X buffer for 30 cycles. Each cycle consists of a denaturation step (95 &#176;C for 30 sec), an annealing step (45 &#176;C for 30 sec) and an extension step (72 &#176;C for 3 min).</li>
	<li>Extract the amplified DNA product with equal volume of phenol/ chloroform (1:1). 
	Note: Phenol is combustible and can cause burns. Chloroform is hazardous. Avoid direct contact with them and use them under a chemical fume hood. Alternatively, PCR products can be purified by various kits.
	<ol>
		<li>Spin at 18,000 x g in a microcentrifuge at room temperature for 1 min. Transfer the aqueous solution to a new tube and extract with an equal volume of chloroform.</li>
		<li>Spin at 18,000 x g in a microcentrifuge at room temperature for 0.5 min. Transfer the aqueous solution to a new tube.</li>
		<li>Mix the aqueous solution with one tenth volume of 3 M Na-acetate and two volumes of absolute ethanol.</li>
		<li>Spin at 18,000 x g in a microcentrifuge at room temperature for 5 min. Discard the aqueous solution.</li>
		<li>Wash the DNA pellet with two volume of 75% ethanol. Discard the aqueous solution and air dry the DNA pellet at room temperature for 5 min. Resuspend the DNA pellet in 34 &#956;l water.</li>
	</ol>
	</li>
	<li>Digest the amplified products in a final volume of 40 &#956;l with restriction enzymes SalI (10 units) and BamHI (10 units) and 4 &#956;l of their 10X buffer at 37 &#176;C for two hr. Resolve the digested products in an agarose gel (1.0% at 100 V) containing ethidium bromide (0.2 &#956;g/ml). 
	Note: Ethidium bromide is a potent mutagen and can be absorbed through skin. Therefore, it is important to avoid direct or indirect contact with ethidium bromide.</li>
	<li>Cut out the correct bands for purification by columns. Weigh the agarose gel containing the DNA fragment. Then dissolve it in the solubilization buffer (300 &#956;l for 0.1 g) at 37 &#176;C for about 20 min.</li>
	<li>Add the resulted solution to a column and spin in a microcentrifuge at room temperature for 5 sec. Discard the flow-through.</li>
	<li>Wash the column with 0.5 ml solubilization buffer by repeating the step 1.5. Wash the column again with 0.75 ml wash buffer by repeating the step 1.5. Spin the column for 2 min to get rid of the remaining wash buffer.</li>
	<li>Elute the DNA fragment with 50 &#956;l water by repeating step 1.5. Estimate the concentration of the DNA fragment by running an aliquot of it against a DNA size ladder in an agarose gel (1.0% at 100 V) containing ethidium bromide (0.2 &#956;g/ml).</li>
	<li>Ligate the purified products (about 100 ng) with the ptdTomato-C1 vector17 (about 100 ng) previously digested with the same restriction enzymes in 10 &#956;l using T4 DNA ligase (2,000 units) and 1 &#956;l 10X ligation buffer at room temperature for 16 hr. 
	Note: The wild-type and motorless mutant contains amino acids from 2 to 963 and 584 to 963, respectively. Previously, a larger motorless KIF5B mutant was used to identify its function9. A smaller motorless KIF5B mutant was used for the present studies to increase its expression levels.</li>
</ol>
2. Immunofluorescence Microscopy
<ol>
	<li>For live-cell or indirect fluorescence imaging with magnification at or below 40X, seed 0.2-0.3 million HeLa cells in 1 ml complete medium [Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS)] in each well of a six-well plate. Grow at 37 &#176;C with 5 % CO2.</li>
	<li>For indirect immunofluorescence studies with magnifications above 40X, wash cover glasses (18 mm x 18 mm; 1.5 thickness) in absolute ethanol briefly and air-dry them in the wells of a six-well plate, inside a biosafety cabinet to avoid contamination.
	<ol>
		<li>Seed 0.2-0.3 million cells in 1 ml complete DMEM medium in each well of a six-well plate.</li>
	</ol>
	</li>
	<li>Preparation of Transfection Complex
	<ol>
		<li>The next day, inside a biosafety cabinet, dilute 0.6 &#181;g (total) of the expression plasmid for tdTomato-tagged wild-type or motorless KIF5B in the presence or absence of an expression plasmid for a cargo candidate protein (pTagCFP-c-MYC5) with 0.1 ml transfection medium in a 1.6 ml microcentrifuge tube. 
		Note: The vector pTagCFP-c-MYC expresses the TagCFP-tagged c-MYC.</li>
		<li>Add 1.8 &#181;l of the transfection reagent to 0.1 ml transfection medium in another 1.6 ml microcentrifuge tube. Typically, prepare a master mix of diluted transfection reagent enough for 12 samples.</li>
		<li>Incubate for 5 min, at room temperature.</li>
		<li>Add the diluted 0.1 ml transfection reagent to the diluted 0.1 ml DNAs. Mix the contents by inverting the tubes. Spin the tubes for 5 sec in a microcentrifuge.</li>
		<li>Incubate for 45 min, at room temperature.</li>
	</ol>
	</li>
	<li>Washing of Cells
	<ol>
		<li>Wash the seeded cells three times with phosphate buffered saline (PBS) during the incubation period for the formation of the transfection complex.</li>
		<li>Replace the medium of the seeded cells in each well with 0.8 ml pre-warmed transfection medium. Return the plates to the incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Transfection of Cells
	<ol>
		<li>After incubation of 45 min, add 0.2 ml of the DNA/transfection reagent complex (prepared in step 2.3) dropwise to each well of the plate.</li>
		<li>Rock the 6 well plate gently for 5 sec, before they are returned to the incubator at 37 &#176;C.</li>
		<li>After 6-8 hr, replace the medium with the complete DMEM medium.</li>
	</ol>
	</li>
	<li>For Live-cell Fluorescence Imaging18
	<ol>
		<li>After an additional 16 hr of incubation at 37 &#176;C, add the DNA intercalating stain Hoechst 33342 to the medium to a final concentration of 1 &#181;M. 
		Note: Hoechst 33342 is a cell-permeable, DNA intercalating dye that stains the cell nuclei.</li>
		<li>Incubate the transfected cells for 10 min in the incubator at 37 &#176;C before examining them for the expression of fluorescent proteins by fluorescent microscopy using a 40X objective. The filter sets for DAPI, CFP, FITC and Cy3 are (Ex350 nm/ Em460 nm), (Ex436 nm/ Em480 nm), (Ex470 nm/ Em525 nm) and (Ex545 nm/ Em605 nm), respectively. 
		Note: If the background is high due to auto-fluorescence of the complete DMEM medium, medium without phenol red is used.</li>
	</ol>
	</li>
</ol>
3. For Indirect Immunofluorescence Studies
<ol>
	<li>Preparation of Paraformaldehyde Solution
	<ol>
		<li>Weigh paraformaldehyde powder (4 g per 100 ml PBS) and add it to PBS. 
		Note: Paraformaldehyde is a flammable solid and a potential cancer hazard. It irritates eyes, respiratory system and skin. Therefore, it is important to avoid contact with or inhalation of paraformaldehyde.</li>
		<li>Add NaOH to the solution (150 &#181;l 10 N NaOH/100 ml).</li>
		<li>Keep the solution at 37 to 42 &#176;C for 2-3 hr with occasional shaking.</li>
		<li>Adjust pH to 7.0 by adding glacial acetic acid to the solution (about 75 &#181;l/ 100 ml) after the paraformaldehyde powder is dissolved.</li>
	</ol>
	</li>
	<li>Target Proteins Staining
	<ol>
		<li>After further incubation for 16 hr at 37 &#176;C, wash the transfected cells at room temperature once with PBS by swirling the six-well plate for 5 sec.</li>
		<li>Fix the cells with 1 ml of freshly prepared, 4% paraformaldehyde solution per well. Incubate at room temperature for 30 min.</li>
		<li>Wash the cells with PBS once by swirling for 5 sec the six-well plate. Discard the solution.</li>
		<li>Incubate the cells with 1 ml of 0.1% Triton-X 100 (in PBS) at room temperature for 30 min. The detergent Triton-X 100 will permeabilize the cell membrane to allow access of antibodies to their intracellular targets.</li>
		<li>Wash the cells with PBS four times by swirling the six-well plate for 5 sec each time. Discard the solution after each wash.</li>
		<li>Incubate the cells with 1 ml of primary antibodies (c-MYC rabbit antibody or p53 mouse antibody; 0.1 &#181;g/ml) in a 10% FBS in PBS solution at room temperature by rocking for 4 hr.</li>
		<li>Wash with PBS four times by swirling the six-well plate for 5 sec each time. Discard the solution after each wash.</li>
		<li>Incubate with 1 ml of fluorescent dye-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit or anti-mouse IgG antibody; 0.5 &#181;g/ml) in 10% FBS in PBS at room temperature in the dark by rocking for 2 hr.</li>
		<li>Wash with PBS four times by swirling the six-well plate for 5 sec each time. Discard the solution after each wash.</li>
	</ol>
	</li>
	<li>Nuclear Staining and Mounting
	<ol>
		<li>Incubate the cells with the 1 ml of DNA intercalating dye 4&#39;,6-diamidino-2-phenylindole (DAPI; 0.5 &#181;g/ml) in PBS at room temperature, in dark for 10 min. Proceed to step 3.4 when no cover glasses are utilized. 
		Note: DAPI is a DNA intercalating dye that stains the cell nuclei.</li>
		<li>Apply 10 &#956;l of mounting solution onto each microscope slide. Mount each cover glass over the mounting solution on a microscope slide.</li>
		<li>Incubate at room temperature in the dark overnight.</li>
		<li>Seal the edges of the cover glasses with nail polish. Place in the dark inside a fume hood overnight to remove the nail polish fumes.</li>
	</ol>
	</li>
	<li>Fluorescence Microscopy19
	<ol>
		<li>Next day, examine the cells by fluorescent microscopy using a 40X objective. The filter sets for DAPI, CFP, FITC and Cy3 are (Ex350 nm/ Em460 nm), (Ex436 nm/ Em480 nm), (Ex470 nm/ Em525 nm) and (Ex545 nm/ Em605 nm), respectively.</li>
	</ol>
	</li>
</ol>

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Free Access publication and production of this article is paid for by Roche.

The method described utilizes the properties of the motorless KIF5B mutant, which lacks the ability to move along the microtubules, but retains the ability to form dimers with the wild-type KIF5B and, thereby, allow the tetrameric protein to interact with the same cargo proteins as the wild-type KIF5B. Motorless KIF5B, therefore, acts as a dominant negative mutant and forms mislocalized aggregates with its cargos. This method is proven to identify the Kinesin-1 cargo c-MYC (Figure 1)5. In this article, the same motorless KIF5B mutant was used to identify p53 as another potential cargo of Kinesin-1 (Figure 2). This shows that the methodology is feasible to identify other cargos of Kinesin-1. Furthermore, the specificity of the mutant is provided by the lack of effect of the mutant on the negative control protein c-Fos (Figure 3).
In this protocol, the tdTomato-tagged wild-type or motorless KIF5B protein is coexpressed with another fluorescent protein-tagged candidate cargo protein. In this case, live-cell fluorescence microscopy and imaging are performed. The formation of the aggregates can be traced by time-lapse imaging. Alternatively, the tdTomato-tagged protein is expressed alone and the candidate cargo protein at its physiological levels is visualized by indirect immunofluorescence microscopy using specific antibodies. The fluorescent protein tdTomato is chosen for its brightness and photostability17. If the background is high due to auto-fluorescence of the complete DMEM medium, medium without phenol red is used.
The motorless KIF5B mutant forms dimers with the wild-type KIF5B stoichiometrically. Therefore, it is critical to express sufficient amount of motorless KIF5B mutant to form dimers with the wild-type counterpart to inhibit its function and to form aggregates. To address this issue, optimization of expression of the motorless mutant is essential. It is achieved by using a small motorless mutant containing the dimerization domain. In addition, optimization of the appropriate transfection reagent and protocol is also necessary. The duration of incubation of HeLa cells with the DNA/ transfection reagent was optimized in HeLa cells for expression of exogenous proteins and cell viability. The incubation duration may have to be optimized for other cell lines.
For magnifications between 4X to 40X, no cover glasses are required. Cells can be directly examined in the wells. Therefore, in this case, the protocol is inexpensive and convenient. For magnifications above 40X, cells are grown on cover glasses in the wells and, after staining, are mounted on microscope slides for examination under oil-immersion objectives.
Observation of aggregates of the motorless KIF5B protein is limited by the size of the cytoplasm. It is easy to detect the aggregates in many mammalian cells. However, it is relatively difficult to observe the aggregates in neuronal cells when the volume of the cytoplasm is small around the nuclei and in neurites.
The protocol is used to show the association between KIF5B and its cargos in addition to the in vivo yeast two-hybrid and biochemical pull-down assays13-16. All these assays determine the association under different physical conditions and their results can complement each other. Moreover, the advantage of this fluorescence assay over other assays is that it can show the regulation of subcellular localization of the cargos by KIF5B (Figures 1 and 2).
The protocol is not limited to KIF5B and can be used to identify cargos of other motor proteins, such as some other kinesin motors3 and some of the myosin motors23,24 used in intracellular transport of cargos3. These motor proteins also contain motor domains for movement along microtubules for kinesin motors and microfilaments for myosin motors, and coiled-coil segments for oligomerization. Most of them form homodimers3,23. Therefore, a similar strategy can be applied to them by creating their motorless dominant negative mutants to identify their cargos.

Kinesin-1 is a motor protein that mediates anterograde transport of its cargos1,2. It is a heterotetramer of the two subunits of Kinesin Light Chain 1 (KLC1) and two subunits of Kinesin Heavy Chains (KHCs). KIF5B1, a KHC, contains a motor domain at its N-terminal, which hydrolyzes ATP and converts the chemical energy to mechanical energy for movement along microtubules. Its C-terminal region contains the dimerization domain that interacts with KLC1, which associates with cargos. Kinesin-1 transports cargos such as vesicles, organelles and mRNAs3,4. Recently, KIF5B was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm5. Three methodologies (chemical inhibitor, siRNA/ shRNA and dominant negative mutant) were used to inhibit Kinesin-1 function. They all induced aggregation of c-MYC in the cytoplasm. For the last methodology, c-MYC was only affected by the dominant negative mutant of KIF5B, but not by that of another related KIF5A motor protein, suggesting that the mutant does not exert general effects on the intracellular components (like microtubule disruption) or on protein aggregation. The dominant negative mutant of KIF5B also did not affect another transcription factor, suggesting that it does not exert general effects on transcription factors. Rather, it suggests that the dominant negative mutant exerts specific effects on its cargos.
The use of dominant negative mutants is common in the field of motor proteins. Similar motorless mutants of kinesins and myosins were used previously. They were mainly used to demonstrate the effects of the mutants on the subcellular localizations of their cargos or on cellular functions6-12. Less emphasis was put on the spatial relationship between the mutants and the cargos affected by them. However, in some incidences, the mutants were observed to co-localize with their cargos6,10.
The interaction between KIF5B and its associated proteins was previously confirmed by the in vivo yeast two-hybrid assay and biochemical pull-down assays such as co-immunoprecipitation and in vitro pull-down assays13-16. In this article, an additional visual method using fluorescence microscopy is described to identify KIF5B cargo proteins. The method makes use of a motorless KIF5B mutant that acts as a dominant negative mutant. It aggregates in the cytoplasm and induces aggregation of its cargos.
The tagging of wild-type and motorless KIF5B mutant with the fluorescent protein tdTomato17 enables their visualization by fluorescence microscopy. The tagged KIF5B proteins can be co-expressed with a candidate protein fused to a different fluorescent protein with spectral properties suitably separated from the KIF5B tag. The tagged proteins are observed directly in live cells under fluorescence microscopy. Induction of aggregation of the candidate protein by the motorless KIF5B mutant will confirm that the candidate protein is an in vivo cargo of KIF5B. Furthermore, the tdTomato-tagged KIF5B proteins can be expressed alone in the cells to study their effects on the endogenous cargo proteins. Later, immunofluorescence microscopy is conducted in which the transfected cells are fixed and stained with a specific antibody against the endogenous candidate protein, followed by an appropriate secondary antibody conjugated with a fluorescent dye. In this case, the endogenous candidate protein at its physiological level is studied. Similar motorless mutants of other motor proteins can be prepared to identify their cargos.

<table><tbody><tr><td>absolute ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>Opti-MEM-I (transfection medium)</td><td>Life Technologies</td><td>51985</td><td></td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Life Technologies</td><td>P36961</td><td></td></tr><tr><td>formaldehyde, para</td><td>Fisher Scientific</td><td>O4042-500</td><td></td></tr><tr><td>Triton-X100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>X-tremeGENE 9 DNA Transfection Reagent</td><td>Roche</td><td>6365787001</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>Life Technologies</td><td>10342020</td><td></td></tr><tr><td>PCR Grade Nucleotide Mix (dNTP Mix)</td><td>Roche</td><td>12111424</td><td></td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12-541A</td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA ladder</td><td>Life Technologies</td><td>SM1333</td><td></td></tr><tr><td>PureLink Quick Gel Extraction kit</td><td>Life Technologies</td><td>K210012</td><td></td></tr><tr><td>BamHI</td><td>New England Biolab</td><td>R0136</td><td></td></tr><tr><td>SalI</td><td>New England Biolab</td><td>R0138</td><td></td></tr><tr><td>T4 DNA ligase&amp;nbsp;</td><td>New England Biolab</td><td>M0202T</td><td></td></tr><tr><td>Ethidium bromide</td><td>Thermo Scientific</td><td>17898</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>c-MYC rabbit antibody</td><td>Cell Signaling</td><td>5606</td><td></td></tr><tr><td>p53 mouse antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated anti-rabbit IgG antibody</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated anti-mouse IgG antibody</td><td>Life Technologies</td><td>A11059</td><td></td></tr><tr><td>fluorescent microscope&amp;nbsp;</td><td>Olympus IX71_Fluoview</td><td></td><td></td></tr><tr><td>computer software for imaging</td><td>&amp;nbsp;cellSens&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

identification of kinesin-1 cargos using fluorescence microscopy

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

Exogenously expressed wild-type KIF5B appeared homogeneously in the cytoplasm, while c-MYC appeared mainly in the nucleus (Figure 1A). However, the motorless KIF5B mutant formed aggregates in the cytoplasm (Figure 1A). Aggregation of the motorless KIF5B induced the aggregation of c-MYC. The percentage of cells expressing aggregated TagCFP-tagged c-MYC in the cells expressing wild-type KIF5B was low. However, it was significantly higher when the motorless KIF5B was co-expressed (Figure 1A). The observed co-localization of mutant KIF5B with c-MYC (Figure 1) suggests that KIF5B regulates the subcellular localization of c-MYC and c-MYC is a cargo of Kinesin-1. Negative controls in which the constructs were expressed alone are included in the lower panel of Figure 1A. The results show that there was no significant bleed-through of fluorescence emission. The motorless KIF5B also induced aggregation of the endogenous c-MYC (Figure 1B) and the transcription factor p53 (Figure 2), indicating that c-MYC and p53 are both the cargos of Kinesin-1 and KIF5B regulates the subcellular localization of both endogenous proteins. Together with the results that the Kinesin-1 inhibitor rose bengal lactone (RBL)20 induces formation of high molecular weight species for both c-MYC and p535, p53 is likely a cargo of Kinesin-1. It is interesting to note that the nuclear transcription factor p53, like c-MYC, is also exported from the nucleus to the cytoplasm for proteasomal degradation21,22. The movement of transcription factors by KIF5B appears to be specific because the expression of the motorless KIF5B mutant did not affect the subcellular localization c-Fos or cause it to aggregate (Figure 3). The above data demonstrate that the method employed in this publication allows for the high-specificity identification of Kinesin-1 cargos. A similar strategy may be applied to other motor proteins as well.
<img alt="Figure 1" src="/files/ftp_upload/53632/53632fig1.jpg" /> 
Figure 1: Expression of the motorless KIF5B mutant induces c-MYC aggregation in the cytoplasm. (A) HeLa cells were transfected with tdTomato-tagged wild-type (WT) KIF5B (red) and TagCFP-tagged c-MYC (blue). tdTomato-tagged WT KIF5B appeared mainly in the cytoplasm, while TagCFP-tagged c-MYC appeared mainly in the nucleus. However, the tdTomato-tagged motorless KIF5B mutant (red) formed filamentous or punctate aggregates in the cytoplasm. Expression of the motorless KIF5B induced the aggregation of c-MYC. The mutant KIF5B and c-MYC co-localized together (pink). Percentages of cells (%) indicating aggregates of CFP-c-MYC (%) are shown. The results are shown as mean&#177;SD (N = 3). Negative controls with expression of tdTomato-tagged KIF5B proteins or TagCFP-tagged c-MYC along with empty vectors (V) are shown in the lower panel. (B) Similar results were obtained with the endogenous c-MYC (green) when tdTomato-tagged WT or motorless KIF5B proteins (red) were expressed. The tdTomato-tagged motorless KIF5B mutant formed aggregates in the cytoplasm and induced aggregation of the endogenous c-MYC. Both kinds of aggregates co-localized together in the cytoplasm (orange). The nuclei were stained with the dye Hoechst 33342 or DAPI. Scale bar; 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/53632/53632fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53632/53632fig2.jpg" /> 
Figure 2: Expression of the motorless KIF5B mutant induces endogenous p53 aggregation in the cytoplasm. In HeLa cells, exogenous tdTomato-tagged wild-type (WT) KIF5B (red) appeared mainly in the cytoplasm, while the endogenous p53 (green) appeared mainly in the nucleus. In contrast, the tdTomato-tagged motorless KIF5B mutant (red) formed aggregates in the cytoplasm. Expression of the motorless KIF5B induced the aggregation of p53, resulting in the co-localization of KIF5B and p53 in the cytoplasm (yellow). The experiment was performed three times. The nuclei were stained with the dye DAPI. Scale bar; 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/53632/53632fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53632/53632fig3.jpg" /> 
Figure 3: Expression of the motorless KIF5B mutant does not induce aggregation of c-Fos in the cytoplasm. tdTomato-tagged wild-type (WT) KIF5B (red) appeared mainly in the cytoplasm of HeLa cells, while EGFP-c-Fos (green) appeared mainly in the nucleus. On the contrary, the tdTomato-tagged motorless KIF5B mutant (red) formed aggregates in the cytoplasm. Expression of the motorless KIF5B did not induce the aggregation of c-Fos. The experiment was performed four times. The nuclei of the cells were stained with the dye DAPI. Scale bar; 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/53632/53632fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>common reverse primer</td>
			<td>5&#8217;-AGAGGATCCTTACACTTGTTTGCCTCCTC-3&#8217;</td>
		</tr>
		<tr>
			<td>wild-type KIF5B forward primer</td>
			<td>5&#8217;- AGAGTCGACGCGGACCTGGCCGAGTGCAACATCAAAGT-3&#8217;</td>
		</tr>
		<tr>
			<td>motorless KIF5B forward primer</td>
			<td>5&#8217;- AGAGTCGACGATGAAGAGTTCACTGTTGC-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences for cloning the wild-type and motorless KIF5B proteins.

Watch this Scientific Journal Video about Identification of Kinesin-1 Cargos Using Fluorescence Microscopy at JoVE.com

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

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

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

identification-of-kinesin-1-cargos-using-fluorescence-microscopy

Research

JoVE Journal

Biology

7.9K Views.  Icahn School of Medicine at Mount Sinai.  Hier wordt een protocol voorgelegd aan Kinesin-1 ladingen te identificeren. Een motorloze mutant van de kinesine-1 zware keten (KIF5B) aggregaten in het cytoplasma en induceert aggregatie van de ladingen. Beide aggregaten gedetecteerd onder fluorescentiemicroscopie. Een soortgelijke strategie kan worden toegepast om ladingen van andere auto eiwitten te identificeren. 

Video: Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Chemistry

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Developmental Biology

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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

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

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

Engineering

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

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

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

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

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

Biochemistry

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

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

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

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

Neuroscience

Cargo Loading onto Kinesin Powered Molecular Shuttles

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

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

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

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

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

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

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

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

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Isolation and Purification of Kinesin from Drosophila Embryos

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

Isolation and Purification of Kinesin from Drosophila Embryos

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

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

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

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