This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (BES-MSE). Kinesin synthesis and fluorescence microscopy were performed through a user project (ZIM) at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science.

1. Preparation of stock microtubule solutions
CAUTION: Safety goggles, gloves and a lab coat should always be worn throughout the protocol.
<ol>
	<li>Prepare 5x BRB80 buffer: add 24.19 g of PIPES (piperazine-N,N&#8242;-bis[2-ethanesulfonic acid]) and 0.38 g of EGTA (ethylene glycol-bis[&#946;-aminoethyl ether]-N,N,N&#8242;,N&#8242;-tetraacetic acid) to a 1 L glass bottle. Add 1 mL of 1 M MgCl2 and adjust the pH to 6.9 with KOH. Add deionized water to bring the solution to a 500 mL final volume.</li>
	<li>Prepare 100 mM stock of GTP solution: weigh 52 mg of GTP and suspend in 1 mL of distilled water. Divide 100 mM solution into 20 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare GPEM solution: mix 200 &#181;L of 5x BRB80, 10 &#181;L of 100 mM GTP solution, 100 &#181;L of 100% glycerol, and 600 &#181;L of deionized water. Divide GPEM solution in 100 &#181;L aliquots and store in -20 &#176;C.&#160;</li>
	<li>Prepare microtubule solution by reconstituting vials of commercially available, lyophilized tubulin (one vial each of biotinylated, fluorescently labeled, and unlabeled) in cold (4 &#176;C) GPEM solution to a stock concentration of 5 mg/mL.</li>
	<li>Perform microtubule polymerization by mixing 4 &#181;L of biotinylated tubulin, 4 &#181;L of fluorescently labeled tubulin, and 24 &#181;L of unlabeled tubulin (all at 5 mg/mL concentrations) to create a ratio of 1:1:6 at a final volume of 32 mL. Keep it on ice. Divide the tubulin mixture into 2 &#181;L aliquots and store at -80 &#176;C until needed. 
	NOTE: Efficient polymerization requires that the concentration of tubulin is equal to or above the critical concentration (5 mg/mL)14. Here, selection of the tubulin ratio is optimized for a sufficient concentration biotinylated tubulin to efficiently bind streptavidin and GUVs, as well as a sufficient concentration of fluorescent tubulin for microscopic characterization.</li>
</ol>
2. Preparation of giant unilamellar vesicles (GUVs)
<ol>
	<li>Agarose film preparation 
	NOTE: This protocol is adapted from Greene et al.15.
	<ol>
		<li>Prepare a 1% w/v solution by mixing 1 g of agarose in 100 mL of deionized water in 250 mL Erlenmeyer flask. Use a standard microwave to heat the agarose solution for 1&#8211;2 min. 
		NOTE: The solution will become translucent once the agarose is completely dissolved. Allow the solution to cool to 65&#8211;75 &#176;C before use.</li>
		<li>Use a cut 1,000 &#181;L pipette tip to pipette 300&#8211;400 &#181;L of agarose solution onto a 25 mm x 25 mm glass coverslip. While holding the edge of the coverslip with gloved fingers, use another 1,000 &#181;L pipette tip to spread the melted agarose evenly across the coverslip. 
		NOTE: Maintaining the agarose at 65&#8211;75 &#176;C will allow for efficient spreading on the coverslip surface.</li>
		<li>Dry the agarose-coated coverslips in a 37 &#176;C incubator for at least 2 h, at which point the agarose will become transparent. Store the coverslips by placing the agarose coated surface facing upwards on a clean surface such as lint-free paper or wax-based film at room temperature (RT).</li>
	</ol>
	</li>
	<li>Lipid formulation
	<ol>
		<li>Retrieve lipids dissolved in chloroform from a -20 &#176;C freezer and place them in a chemical fume hood until they reach RT.</li>
		<li>Calculate the volume of lipid stock of each component lipid needed for the formulation using following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/60899/60899eq1.jpg" /> 
		Where: Vlipid is the volume of lipid to use, mol % is the molar percentage of the lipid component, n is the total number of moles of lipid used in the formulation, and M is the concentration of lipid in molar units. 
		NOTE: For example, if using a formulation containing 45 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with a stock solution concentration of 12.72 mM, and 1 micromole of total lipids in the formulation, the volume of DOPC stock used in the formulation would be: 
		<img alt="Equation 2" src="/files/ftp_upload/60899/60899eq2.jpg" /></li>
		<li>Mix the lipids together at the calculated ratio in a glass vial in the chemical fume hood.</li>
		<li>Pipette 30 &#181;L of lipid solution onto the agarose-coated coverslips on a preheated hot plate that is set above the melting point of the saturated lipid component of the formulation (e.g., 50 &#176;C hot plate for a lipid with Tm of 40 &#176;C).</li>
		<li>Spread the solution across the agarose film in a circular motion using the long edge of an 18 G needle to until the chloroform has evaporated and a uniform layer of lipid has formed. Hold the edge of the coverslip with gloved fingers while performing this step. 
		NOTE: Care should be taken not to damage the agarose layer with the needle.</li>
		<li>Place the coverslip with agarose layer and lipid film in an aluminum foil-covered Petri dish, film side facing upwards, and place the Petri dish in a vacuum desiccator for at least 2 h to remove residual solvent.</li>
	</ol>
	</li>
	<li>In the meantime, prepare 560 mM sucrose solution by mixing 1.92 g of sucrose with 10 mL of deionized water. 
	NOTE: The concentration of the sucrose solution depends on the osmolarity of the buffer GUVs are diluted in. Typically, the osmolarity of the sucrose solution should be no more than 10% larger than the buffer the GUVs will be diluted in, specifically the motility buffer (see step 3.11).</li>
	<li>GUV formation
	<ol>
		<li>Adhere an adhesive chamber to the coverslip coated with the lipid film by gently pressing the adhesive chamber onto the lipid-coated coverslip with the lipid film facing upwards, ensuring that a tight seal is formed.</li>
		<li>Add 400 &#181;L of 560 mM sucrose solution (prepared in step 2.3) to the chamber.&#160;</li>
		<li>Place the coverslip into the humidity chamber and close the lid.</li>
		<li>Place the humidity chamber on a preheated hot plate set above the melting point of the saturated lipid component of the formulation.</li>
		<li>Allow vesicles to form for &#8805;1 h before recovery. 
		NOTE: Vesicle formation may be checked with fluorescence microscopy with a 40x air objective lens.</li>
	</ol>
	</li>
</ol>
3. Preparation of motility assay stocks and reagents
<ol>
	<li>Preparation of casein stock
	<ol>
		<li>Add 3 g of dry casein to a 50 mL conical centrifuge tube, then add 30 mL of 1x BRB80 and rotate until the solution becomes viscous. Centrifuge the tube at 15,000 x g for 30 min.</li>
		<li>Transfer the supernatant into another 50 mL conical centrifuge tube and discard the pellet. Filter the solution through a 1 &#181;m syringe filter, collecting the solution in a 50 mL conical vial. Filter the solution through a 0.2 &#181;m filter, collecting the solution in a 50 mL conical vial.</li>
		<li>Determine the protein concentration by measuring the absorbance at 280 nm using a UV-Vis spectrophotometer and quartz cuvette.</li>
		<li>Calculate the casein concentration in mg/mL using the following formula: 
		<img alt="Equation 3" src="/files/ftp_upload/60899/60899eq3.jpg" /></li>
		<li>Dilute the solution to 20 mg/mL in 1x BRB80, divide into 100 &#181;L aliquots, and store at&#160;-20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare glucose oxidase (2 mg/mL) stock solution by mixing 2 mg of glucose oxidase with 1 mL of 1x BRB80. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare catalase (0.8 mg/mL) stock solution by mixing 0.8 mg of catalase with 1 mL of 1x BRB80. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 2 M glucose solution by suspending 0.3 g of D-glucose in 1 mL of deionized water. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM DTT stock by suspending 0.015 g of DTT in 1 mL of deionized water. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Mg-ATP stock by suspending 0.055 g of disodium ATP in 1 mL solution of 100 mM MgCl2. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Mg-AMP-PNP solution by suspending out 0.055 g of AMP-PNP in a 1 mL solution of 100 mM MgCl2, then divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Trolox solution by adding 25.03 mg of Trolox to 1 mL of methanol and store at -20 &#176;C.</li>
	<li>Prepare 10 mg/mL streptavidin solution by adding 1 mg of streptavidin to 100 &#181;L of BRB80, then divide into 2 &#181;L aliquots and and store at -80&#1755; &#176;C.</li>
	<li>Prepare BRB90CAT by mixing 200 &#181;L of 5x BRB80, 20 &#181;L of casein solution, 10 &#181;L of MgATP solution, 10 &#181;L of Trolox, 5 &#181;L of paclitaxel solution, and 765 &#181;L of DI water. Store at 4 &#176;C.</li>
	<li>Prepare the motility solution by mixing 192 &#181;L of BRB80CAT, 2 &#181;L of D-glucose solution, 2 &#181;L of glucose oxidase solution, 2 &#181;L of DTT solution, and 2 &#181;L of catalase solution. Store at 4 &#176;C.</li>
	<li>Prepare a 1 &#181;M kinesin solution by diluting the stock kinesin solution in BRB80CAT (e.g., 2 &#181;L of 50 mM kinesin solution in 98 &#181;L of BRB80CAT) and store at 4 &#176;C. &#160;</li>
	<li>Prepare a 10 &#181;g/mL microtubule solution by diluting 10 &#181;L of stabilized microtubules in 90 &#181;L of room temperature BRB80CAT. Store at RT.</li>
	<li>Prepare a 10 &#181;g/mL streptavidin solution by adding 0.1 &#181;L of 10 mg/mL streptavidin solution in 99.9 &#181;L of motility solution. Store at 4 &#176;C.</li>
	<li>Prepare a 12x GUV solution by diluting 5 &#181;L of GUV stock into 55 &#181;L of motility solution. Store at 4 &#176;C.</li>
	<li>Polymerization of tubulin into microtubules
	<ol>
		<li>Collect a previously prepared 2 &#181;L aliquot of tubulin from the -80 &#176;C freezer (prepared in step 1.5) and place into a 37 &#176;C water bath for 30 min.</li>
		<li>Prepare a 2 mM paclitaxel solution by adding 1.71 mg of paclitaxel in 1 mL of anhydrous DMSO, divide into 10 &#181;L aliquots, and store at -20 &#176;C.</li>
		<li>Freshly prepare BRB80T by mixing 99.5 &#181;L of 1x BRB80 with 0.5 &#181;L of 2 mM paclitaxel. &#160;Warm 100 &#181;L of BRB80T to 37 &#176;C in the water bath.</li>
		<li>After 30 min, add the 100 &#181;L of BRB80T to the tubulin aliquot to stabilize the microtubules. Store at RT.</li>
	</ol>
	</li>
</ol>
4. Gliding motility assay (GMA)
<ol>
	<li>Prepare a flow chamber by affixing two strips of double-sided tape separated by 5 mm onto a glass slide. Repeat this process until three layers of tape comprise each strip.</li>
	<li>Place a coverslip on top of the tape, then press down gently on the coverslip/tape interface with a tweezer or pen to ensure sufficient adhesion. 
	NOTE: The channel should be 5 mm in width by 25 mm in length by 300 mm in height.</li>
	<li>Pipette 30 &#181;L of 1 mm kinesin solution (prepared in step 3.12) into the flow cell and let it incubate for 5 min. 
	NOTE: The casein forms a bilayer on the surface of the coverslip/glass slide and facilitates attachment of the kinesin tail to the surface.</li>
	<li>Pipette 30 &#181;L of 10 &#181;g/mL microtubule solution (prepared in step 3.13) into the flow cell, using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 5 min.</li>
	<li>Wash the flow cell 1x&#8211;3x with 1x motility solution at RT (prepared in step 3.11). &#160; 
	NOTE: Fluorescence microscopy using a 40x air objective may be used at this point to confirm microtubule attachment and motility. Microtubules appear as fluorescent filaments (tens of microns in length) moving (gliding) across the surface at ~0.5 &#956;m/s (Figure 1).</li>
	<li>Pipette 30 &#181;L of 10 &#181;g/mL streptavidin solution (prepared in step 3.14) into the flow cell using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 10 min.</li>
	<li>Flow 30 &#181;L of 12x GUV solution (prepared in step 3.15) into the flow using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 30 min.</li>
	<li>Add 2 &#181;L of 100 mM AMP-PNP solution (prepared in step 3.7) to stop motility, then seal the chamber with sealant.</li>
</ol>
5. LNT network characterization
<ol>
	<li>Transfer the flow chamber to an inverted microscope for imaging.</li>
	<li>Choose the appropriate filter set based on the wavelengths of the fluorescent lipids or tubulin used. For example, when using Texas Red-labeled lipids, use a 560 nm/25 nm excitation filter and 607 nm/36 nm emission filter.</li>
	<li>Use a 100x oil objective to focus on the surface of the coverslip.</li>
	<li>Image the LNT networks using fluorescence microscopy. 
	NOTE: LNTs are linear structures extruding from the larger vesicles. LNTs much smaller than GUVs and have weaker fluorescence signals. Thus, the exposure and contrast must be adjusted accordingly to image LNTs. These adjustments also lead to overexposure of the GUVs, and thus, it is recommended that the lamellarity and phase separation in GUVs be characterized independently.</li>
	<li>Focus the microscope on a network of interest and take a standard or tiled image.</li>
	<li>Adjust the exposure time and neutral density filters to image the LNTs and minimize saturated exposure of the GUVs. Acquire images both in red and green channels. 
	NOTE: Here, the red channel permits visualization of the Texas-Red lipids, while the green channel permits visualization of the microtubules (e.g., Oregon Green lipids and HiLyte488 dyes).</li>
	<li>Create a composite image by overlaying the red and green channels (Figure 1).</li>
	<li>Characterization of LNT networks by measuring LNT length
	<ol>
		<li>Open the acquired images using an image analysis software such as ImageJ.</li>
		<li>Calibrate the scale for the microscope by using the set scale feature, fill in the pixels to the mm conversion factor, and click OK. 
		NOTE: The conversion factor is dependent on the microscope, objective lens, and camera, and it can be obtained using a microscope calibration slide. It is generally expressed in pixels/mm.</li>
		<li>Use the Multipoint line tool to measure the length of the nanotubes starting from the parent GUV. Hold Ctrl +M to measure the length.</li>
		<li>Continue measuring the lengths of individual tubes following the steps above. The image processing tool will save each new measurement in the results window.</li>
		<li>Hold Ctrl + D after drawing each line to keep track of which tubes have been measured.</li>
	</ol>
	</li>
	<li>Measuring LNT thickness (Figure 2)
	<ol>
		<li>Open the image in ImageJ and select the Threshold feature under the Image tab.</li>
		<li>Click Apply to apply the threshold.</li>
		<li>Draw a rectangle of known length over the desired tube (black pixels have a value of 0, and red pixels have a value of 255).</li>
		<li>Measure the integrated density of the area.</li>
		<li>Divide the density by the length (in pixels) of the LNT to obtain the thickness (in pixels). 
		NOTE: The thickness measurements may only be compared across images when the imaging settings and threshold are set identically.</li>
	</ol>
	</li>
	<li>Determining the lipid partitioning in nodes of LNTs (Figure 3)
	<ol>
		<li>Open the image in ImageJ.</li>
		<li>Use the Line tool to draw a line over the desired node.</li>
		<li>Measure the node intensity in both the Oregon Green and Texas Red channels.</li>
		<li>Move the line to the LNT and measure the LNT intensity in both the Oregon Green and Texas Red channels.</li>
	</ol>
	</li>
</ol>

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Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology &#38; Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. DOE&#8217;s National Nuclear Security Administration under contract DE-NA-0003525. This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

LNT networks are a useful tool for in vitro studies to membrane properties and the transport of biomolecules such as transmembrane proteins. Moreover, using complex lipid formulations to fabricate LNT networks enables more biologically relevant studies. Other fabrication studies have used either 1) simple lipid formulations and multilamellar vesicles or 2) more cumbersome motility techniques to fabricate networks from GUVs comprised of complex lipid formulations. The method described here enables the efficient fabricatio...

The fabrication of lipid nanotube (LNT) networks is of increasing interest for in vitro examination of nonequilibrium lipid structures1,2,3. Cells use lipid tubules for the diffusive transport of proteins4 and nucleic acids5 as well as cell-to-cell communication6,7. The endoplasmic reticulum and Golgi apparatus are particularly interesting, as these membrane-bound organelles are the primary locations for lipid and protein synthesis as well as transport of these integral biomolecules within the cytoplasm of a cell8,9. The membranes of these organelles are comprised of multiple lipid species including sphingolipids, cholesterol, and phospholipids10 that ultimately help define their functionality. Thus, to more closely replicate and study these organelles, in vitro LNTs must be fabricated from vesicles with increasingly complex lipid formulations11.
Giant unilamellar vesicles (GUVs) are used pervasively for studying lipid membrane behavior because they can be reliably synthesized with complex formulations that include cholesterol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)12,13. Described here is a method to fabricate LNTs from GUVs with varying lipid formulations using the gliding motility assay (GMA), in which LNTs are extruded based on the work performed by kinesin motors and microtubule filaments acting on GUVs. In this system, kinesin motor proteins adsorbed to a surface propel biotinylated microtubules, converting chemical energy from the hydrolysis of ATP into useful work (specifically, the extrusion of LNTs from biotinylated vesicles)11. The resulting LNT network provides a model platform to study effects of the differences in lipid phases on changes in LNT morphology.
Briefly, kinesin motor proteins are introduced into a flow chamber in a solution containing casein, which enables the adsorption of the motors onto the glass surface of the chamber. Next, biotinylated microtubules in a solution containing ATP flow through the chamber and are allowed to bind to the kinesin motors and begin motility. A streptavidin solution is then introduced into the chamber and allowed to bind non-covalently to the microtubules. Finally, GUVs containing a biotinylated lipid are introduced into the chamber and bind to the streptavidin-coated microtubules, then extrude LNTs to form large-scale networks over the course of 15&#8211;30 min. This method produces large, branched LNT networks using standard laboratory equipment and reagents at a low cost11.

<table><tbody><tr><td>100x/1.4 Numerical Aperture Oil Immersion Objective</td><td>Olympus</td><td>1-U2B836</td><td>Olympus UPlanSApo 100x/1.40 Oil Objective Infinity Corrected, RMS Thread&lt;br/&gt; Working Distance 0.12mm</td></tr><tr><td>3.0 ND Filter</td><td>Olympus</td><td></td><td>Neutral Density Filter</td></tr><tr><td>AMP-PNP</td><td>Sigma-Aldrich</td><td>A2647</td><td>(&amp;beta;,&amp;gamma;-imidoadenosine 5&amp;prime;-triphosphate)</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A7699</td><td>Adenosine 5&#039;-triphosphate disodium salt hydrate BioXtra</td></tr><tr><td>Brightline Pinkel DA/FI/TR/Cy5/Cy7-5X-A000 filter set</td><td>Semrock</td><td>LED-DA/FI/TR/Cy5/Cy7-5X-A-000</td><td>&lt;br/&gt; BrightLine Pinkel filter set, optimized for DAPI, FITC, TRITC, Cy5 &amp;amp; Cy7 and other like fluorophores, illuminated with LED-based light sources</td></tr><tr><td>Casein</td><td>Sigma-Aldrich</td><td>22090</td><td>Casein hydrolysate for microbiology</td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C9322</td><td>Catalase from Bovine Liver</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306</td><td>Chloroform anhydrous contains 0.5-1.0% ethanol as stabilizer</td></tr><tr><td>Cholesterol</td><td>Avanti</td><td>700000P</td><td>cholesterol (ovine wool, &gt;98%) (powder)</td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>D-(+)-Glucose powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;99.5%</td></tr><tr><td>DOPC</td><td>Avanti</td><td>850375C</td><td>1,2-Dioleoyl-sn-glycero-3-phosphocholine (in chloroform)</td></tr><tr><td>DOPE-Biotin</td><td>Avanti</td><td>870282C</td><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td></tr><tr><td>DPPC</td><td>Avanti</td><td>850355P</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (powder)</td></tr><tr><td>DPPE-Biotin</td><td>Avanti</td><td>870285P</td><td>1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>43816</td><td>DL-Dithiothreitol solution 1 M</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E4378</td><td>EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid</td></tr><tr><td>Glucose Oxidase</td><td>Sigma-Aldrich</td><td>G6125</td><td>Glucose Oxidase from Aspergillus niger&lt;br/&gt; Type II, &amp;ge;10,000 units/g solid (without added oxygen)</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33</td><td>Glycerol (Certified ACS), Fisher Chemical</td></tr><tr><td>GTP</td><td>Sigma-Aldrich</td><td>G8877</td><td>Guanosine 5&amp;prime;-triphosphate sodium salt hydrate</td></tr><tr><td>IX-81 Olympus Microscope</td><td>Olympus</td><td>N/A</td><td>IX81 Inverted Microscope from Olympus</td></tr><tr><td>KOH</td><td>Sigma-Aldrich</td><td>1050121000</td><td>Potassium Hydroxide</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M1028</td><td>1.00 M magnesium chloride solution</td></tr><tr><td>Orca Flash 4.0 Digital Camera</td><td>Hamamatsu</td><td>C13440-20CU</td><td>ORCA-Flash 4.0 V3 Digital CMOS camera</td></tr><tr><td>Oregon Green-DHPE</td><td>Invitrogen</td><td>O12650</td><td>Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine</td></tr><tr><td>Paclitaxel</td><td>ThermoFisher</td><td>P3456</td><td>Paclitaxel (Taxol Equivalent) - for use in research only</td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&amp;prime;-bis(2-ethanesulfonic acid)</td></tr><tr><td>Texas Red-DHPE</td><td>Invitrogen</td><td>T1395MP</td><td>Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine,&lt;br/&gt; Triethylammonium Salt</td></tr><tr><td>Trolox</td><td>Sigma-Aldrich</td><td>238813</td><td>(&amp;plusmn;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td></tr><tr><td>Tubulin, Biotin</td><td>Cytoskeleton</td><td>T333P</td><td>Tubulin protein (biotin) porcine brain</td></tr><tr><td>Tubulin, Hy-Lite 488</td><td>Cytoskeleton</td><td>TL488M</td><td>Tubulin protein (fluorescent HiLyte 488) porcine brain</td></tr><tr><td>Tubulin, Unlabeled</td><td>Cytoskeleton</td><td>T240</td><td>Tubulin protein porcine brain</td></tr></tbody></table>

fabricating multi-component lipid nanotube networks using the gliding kinesin motility assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

LNT networks (Figure 4) were fabricated using the described protocol, which uses the work performed by kinesin transport of microtubules to extrude LNTs from GUVs. Briefly, GUVs were prepared using agarose gel rehydration using sucrose solution, and microtubules were polymerized in GPEM solution and stabilized in BRB80T. Next, kinesin motors were introduced into a flow cell forming an active layer&#160;of motors on the surface of the coverslip. Microtubules were then introduced and a strepta...

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Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

fabricating-multi-component-lipid-nanotube-networks-using-gliding

Research

JoVE Journal

Bioengineering

1.6K Views.  Sandia National Laboratories. Dit protocol maakt het mogelijk om eenvoudig grootschalige lipide nanobuisnetwerken te genereren waarin de individuele nanobuisjes fasescheidingsgedrag vertonen dat vergelijkbaar is met dat van het moederblaasje. Het belangrijkste voordeel van deze aanpak is dat we het werk van de moleculaire motoren coöpteren om de lipide nanobuisjes zelf op een zeer parallelle manier en zonder menselijke tussenkomst te assembleren. Om een glijdende motiliteitstest op te zetten, bevestigt u eerst drie sets ...

Video: Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

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

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Biochemistry

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 ...

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 ...

Biology

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 ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

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