Name: controlling flow speeds of microtubule-based 3d active fluids using temperature
Uploaded: 2019-11-26T14:30:00
Description: Watch this Scientific Journal Video about Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature at JoVE.com

Plasmid K401-BCCP-H6 was a gift from Dr. Zvonimir Dogic. This research was supported by Dr. Kun-Ta Wu's start-up fund in Worcester Polytechnic Institute. We thank Dr. Zvonimir Dogic for the protocols to purify and label tubulin and to synthesize active fluids. We are grateful to Dr. Marc Ridilla for his expertise in protein expression and purification. We thank Dr. William Benjamin Roger for assisting us with building the temperature-controlled stage. We acknowledge Brandeis MRSEC (NSF-MRSEC-1420382) for use of the Biological Materials Facility (BMF). We acknowledge the Royal Society of Chemistry for adapting the figures from Bate et al. on Soft Matter33.

1. Preparation of MTs
CAUTION: In this step we purify tubulins from bovine brain tissue. Bovine brain may cause variant Creutzfeldt-Jakob disease (vCJD)35. Therefore, the brain waste and related solutions, bottles, and pipette tips should be collected in a biowaste bag and disposed of as biohazardous waste according to the rules of the institution.
<ol>
	<li>Purify tubulins from bovine brain (modified from Castoldi et al.36).
	<ol>
		<li>Transp...

Controlling active matter in situ opens the door to directed self-organization of active matter4,5,24,28,54. In this article, we present a protocol for using temperature to control kinesin-driven, MT-based active fluids in situ, based on the Arrhenius characteristic of the system29,30,<sup c...

Active matter is differentiated from conventional passive matter due to its capability to convert chemical energy into mechanical work. A material that possesses such capability can consist of living or non-living entities such as bacteria, insects, colloids, grains, and cytoskeletal filaments1,2,3,4,5,6,7,8,9,10. These material entities interact with their neighbors. At a larger scale, they self-organize into either turbulent-like vortices (active turbulence) or material flows11,12,13,14,15,16,17,18,19,20. An understanding of self-organization of active matter has led to various applications in molecular shuttles, optical devices, and parallel computation21,22,23. To bring applications to the next level requires control beyond self-organization. For example, Palacci et al. developed a hematite-encapsulated colloid that self-propelled only when exposed to manually controlled blue light, which led to the emergence of living crystals24. Morin et al. established the control of rolling Quincke colloids by using a tunable external electric field, resulting in colloidal flocking in a racetrack-like channel25. These previous works demonstrate the role of local control in applications and advance the knowledge base of active matter.
In this article, we focus on the controllability of kinesin-driven, microtubule (MT)-based 3D active fluids. The fluids consist of three main components: MTs, kinesin molecular motors, and depletants. The depletants induce a depletion force to bundle the MTs, which are later bridged by motor clusters. These motors walk along the MTstoward the plus end. When a pair of bridged MTsis antiparallel, the corresponding motors walk in opposite directions. However, the motors are bound in a cluster and are unable to walk apart, so they cooperatively slide apart pairs of MTs (interfilament sliding, Figure 1A). These sliding dynamics accumulate, causing bundles of MTsto extend until reaching their buckling instability point and break (extensile bundles, Figure 1B)26. The broken bundles are annealed by the depletion force, which subsequently extends again, and the dynamics repeat. During the process of the repeating dynamics, the bundle movements stir the nearby liquid, inducing flows that can be visualized by doping with micron-scale tracers (Figure 1C). Sanchez et al. and Henkin et al. have characterized the mean speeds of tracers, finding that the speeds were tunable by varying the concentrations of adenosine triphosphate (ATP), depletants, motor clusters, and MTs19,27. However, such tunability existed only prior to active fluid synthesis. After synthesis, the tunability was lost, and the fluids self-organized in their own way. To control active fluid activity after synthesis, Ross.et al. reported a method using the light-activated dimerization of motor proteins, allowing fluid activity to be tuned on and off using light28. While light control is convenient in terms of locally activating the fluids, the method requires redesigning the structures of motor proteins, along with modifying the optical paths in a microscope. Here, we provide an easy-to-use method for locally controlling fluid flows without microscope modification while keeping the motor structure intact.
Our method of locally tuning active fluid flow is based on the Arrhenius law because the kinesin-MT reaction has been reported to increase with temperature29,30,31,32. Our previous studies showed that the temperature dependence of the mean speed of an active fluid flow followed the Arrhenius equation: v = A exp(-Ea/RT), where A is a pre-exponential factor, R is the gas constant, Ea is the activation energy, and T is the system temperature33. Therefore, fluid activity is sensitive to the temperature environment, and the system temperature needs to be consistent to stabilize the motor performance, and consequently, the fluid flow speed34. In this article, we demonstrate the use of the motor&#39;s temperature dependence to continuously tune the flow speeds of active fluids by adjusting the system temperature. We also demonstrate the preparation of an active fluid sample, followed by mounting the sample on a microscope stage whose temperature is controlled via computer software. Increasing the temperature from 16 &#176;C to 36 &#176;C speeds up the mean flow speeds from 4 to 8 &#181;m/s. Additionally, the tunability is reversible: repeatedly increasing and decreasing the temperature sequentially accelerates and decelerates the flow. The demonstrated method is applicable to a wide range of systems where the main reactions obey the Arrhenius law, such as the MT gliding assay29,30,31,32.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
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			<td>D128</td>
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			<td>Sigma-Aldrich</td>
			<td>E3889</td>
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			<td>18861</td>
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			<td>Glucose Oxidase from Aspergillus niger</td>
			<td>Sigma-Aldrich</td>
			<td>G2133</td>
			<td></td>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
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			<td>GpCpp</td>
			<td>Jena Bioscience</td>
			<td>NU-405L</td>
			<td>Guanosine-5'[(&#945;,&#946;)-methyleno]triphosphate (GMPCPP)</td>
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			<td>GS Power&#39;s 18 Gauge (True American Wire Ga), 100 feet, 99.9% Stranded Oxygen Free Copper OFC, Red/Black 2 Conductor Bonded Zip Cord Power/Speaker Electrical Cable for Car, Audio, Home Theater</td>
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			<td>Sigma-Aldrich</td>
			<td>G8877</td>
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			<td>Sigma-Aldrich</td>
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			<td>I5513</td>
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			<td>I6758</td>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
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			<td>369687</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
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			<td>Lysozyme from chicken egg white</td>
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			<td>L6876</td>
			<td></td>
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			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td>MgCl2&#8226;6H2O</td>
		</tr>
		<tr>
			<td>MES sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>M5057</td>
			<td>2-(N-Morpholino)ethanesulfonic acid sodium salt</td>
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		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td>3-(N-Morpholino)propanesulfonic acid</td>
		</tr>
		<tr>
			<td>MP-3022</td>
			<td>TE Technology</td>
			<td>MP-3022</td>
			<td>Thermocouple</td>
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		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine 99%, ACROS Organics</td>
			<td>Fisher Scientific</td>
			<td>AC138450500</td>
			<td>TEMED</td>
		</tr>
		<tr>
			<td>Nanodrop 2000c UV-VIS Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>E112352</td>
			<td>Spectrometer</td>
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			<td>Nikon</td>
			<td>MEA54000</td>
			<td></td>
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			<td>Norland Products</td>
			<td>NOA81</td>
			<td>UV glue</td>
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		<tr>
			<td>Novex Sharp Pre-stained Protein Standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>LC5800</td>
			<td>Protein standard ladder</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels, 1.5 mm, 10-well</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0335BOX</td>
			<td>SDS gel</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>365672</td>
			<td></td>
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		<tr>
			<td>Parafilm PM996 Wrap , 4&#34; Wide; 125 Ft/Roll</td>
			<td>Cole-Parmer</td>
			<td>EW-06720-40</td>
			<td>Wax film</td>
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		<tr>
			<td>Pe 300 ultra Illumination System Single 
			Band , 3mm Light Guide control Pod 
			power supply</td>
			<td>Nikon</td>
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			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>PMSF</td>
		</tr>
		<tr>
			<td>Phosphoenolpyruvic acid monopotassium salt, 99%</td>
			<td>BeanTown Chemical</td>
			<td>129745</td>
			<td>PEP</td>
		</tr>
		<tr>
			<td>Pierce Coomassie (Bradford) Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Mini Tablets</td>
			<td>Thermo Fisher Scientific</td>
			<td>A32953</td>
			<td></td>
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		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td>1,4-Piperazinediethanesulfonic acid</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
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		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>81300</td>
			<td>PEG. Average molecular weight 20,000 Da</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (Pellets/Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>P250-500</td>
			<td>KOH</td>
		</tr>
		<tr>
			<td>PowerEase 300W Power Supply (115 VAC)</td>
			<td>ThermoFisher Scientific</td>
			<td>PS0300</td>
			<td>DC power supply of the gel box</td>
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		<tr>
			<td>PS-12-8.4A</td>
			<td>TE Technology</td>
			<td>PS-12-8.4A</td>
			<td>DC power supply of the temperature controller</td>
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		<tr>
			<td>Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td>
			<td>Sigma-Aldrich</td>
			<td>P-0294</td>
			<td>PK/LDH</td>
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			<td>Quiet One Lifegard Fountain Pump, 296-Gallon Per Hour</td>
			<td>Amazon</td>
			<td>B005JWA612</td>
			<td>Fish tank pump</td>
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			<td>Rosetta 2(DE3)pLysS Competent Cells - Novagen</td>
			<td>Millipore Sigma</td>
			<td>71403</td>
			<td>Competent cells</td>
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		<tr>
			<td>Sharp Microwave ZSMC0912BS Sharp 900W Countertop Microwave Oven, 0.9 Cubic Foot, Stainless Steel</td>
			<td>Amazon</td>
			<td>B01MT6JZMR</td>
			<td>Microwave for boiling the water</td>
		</tr>
		<tr>
			<td>Sodium Chloride (Crystalline/Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>SDS</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td>NaH2PO4</td>
		</tr>
		<tr>
			<td>Streptavidin Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>21122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
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		<tr>
			<td>TC-720</td>
			<td>TE Technology</td>
			<td>TC-720</td>
			<td>Temperature controller</td>
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		<tr>
			<td>Tris Base, Molecular Biology Grade - CAS 77-86-1 - Calbiochem</td>
			<td>Sigma-Aldrich</td>
			<td>648310</td>
			<td>Tris-HCL</td>
		</tr>
		<tr>
			<td>Type 45 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>339160</td>
			<td></td>
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		<tr>
			<td>Type 70 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
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		<tr>
			<td>Type 70.1 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>342184</td>
			<td></td>
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		<tr>
			<td>VWR General-Purpose Laboratory Labeling Tape</td>
			<td>VWR</td>
			<td>89097-916</td>
			<td>Paper tapes</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glasses, Square, No. 1 1/2</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td>Glass coverslips</td>
		</tr>
		<tr>
			<td>VWR Plain and Frosted Micro Slides, Premium</td>
			<td>VWR</td>
			<td>75799-268</td>
			<td>Glass slides</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell</td>
			<td>ThermoFisher Scientific</td>
			<td>EI0001</td>
			<td>Gel box</td>
		</tr>
		<tr>
			<td>ZYLA 5.5 USB3.0 Camera</td>
			<td>Nikon</td>
			<td>ZYLA5.5-USB3</td>
			<td>Monochrome CCD camera</td>
		</tr>
	</tbody>
</table>

controlling flow speeds of microtubule-based 3d active fluids using temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

Preparing the kinesin-driven, MT-based active fluids requires both kinesin and MTs. The MTs were polymerized from labeled tubulins (steps 1.3 and 1.4) that were purified from bovine brains (step 1.1, Figure 2A), followed by recycling to enhance purity (step 1.2, Figure 2B). The kinesin motor proteins were expressed in and purified from E. coli (steps 2.1 and 2.2, Figure 2B)...

Watch this Scientific Journal Video about Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature at JoVE.com

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

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Fluid Mechanics

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Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Determining 3D Flow Fields via Multi-camera Light Field Imaging

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and ...

High-resolution, High-speed, Three-dimensional Video Imaging with Digital Fringe Projection Techniques

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Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids

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Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding

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Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

Writing and Low-Temperature Characterization of Oxide Nanostructures

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Visualizing Hyporheic Flow Through Bedforms Using Dye Experiments and Simulation

Advective exchange between the pore space of sediments and the overlying water column, called hyporheic exchange in fluvial environments, drives solute ...

Characterization of Full Set Material Constants and Their Temperature Dependence for Piezoelectric Materials Using Resonant Ultrasound Spectroscopy

During the operation of high power electromechanical devices, a temperature rise is unavoidable due to mechanical and electrical losses, causing the ...