Name: membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution
Uploaded: 2016-08-16T13:00:00
Description: Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&amp;D Project) to R.T. and Nanospot GmbH supported this work.

1. Preparation of Large Unilamellar Vesicles (LUVs)
<ol>
	<li>Clean a round bottom flask (10 ml volume) with nitrogen gas to remove any dust particles. Rinse the round bottom flask with ethanol. 
	Note: Residual ethanol can be tolerated and does not disturb subsequent steps.</li>
	<li>Wash a 1 ml glass syringe 4x with chloroform, to remove any contamination. Add 4 ml SoyPC20 (25 mg/ml in chloroform) to the round bottom flask and dope the lipid solution with an additional DOPEATTO390 to a fina...

<ol>
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The technique presented here allows a highly parallel analysis of membrane protein transport. Reconstituted membrane protein systems can directly be applied to the biochip, making the adaption of theoretically every membrane transporter or channel possible. Transport analysis is only limited by the establishment of a fluorescent read-out system, either via direct fluorescence change (translocation of fluorophores or fluorescently labeled substrates) or indirect fluorescence change (pH-sensitive dyes, secondary enzymatic ...

The analysis of membrane proteins has become of increasing interest for basic and pharmaceutical research in the past 20 years. The development of novel drugs depends on the identification and detailed characterization of new targets, currently being one of the limiting factors. The fact that about 60% of all drug targets are membrane proteins1, makes the development of techniques to elucidate their function most important.
In the past, techniques for the study of electrogenic channels and transporters have been developed in multitude2-4. Non-electrogenic substrates in contrary present a more challenging task. They are however of special interest as prime drug targets, as they control the flux of solutes and nutrients across the cell membrane and function as key receptors in signaling cascades5.
Considerable effort has been put into the development of techniques to study the function of membrane transport proteins6,7. Systems using solid-supported membranes have emerged as most promising tools in this field8-10, including solid supported lipid bilayers, tethered bilayers11,12, microblack lipid membranes13,14 and native vesicle arrays15,16 to name a few. Some of them are even available as commercial setups17,18. Some examples have been published combining the ability to study single membrane proteins in a highly parallel manner14,19, a prerequisite for screening applications. However, these methods rarely bridge from basic research to the industrial environment. The difficulties often lie in the ability of the system to be automatable, the cost-intensive production and/or laborious preparation. An approach overcoming all the above mentioned obstacles is the final aim.
The technique presented here was developed to study membrane channels and transporters in vitro in a controlled environment on the single protein level20-22. Reconstitution of purified membrane proteins into LUVs is much more established than comparable approaches for GUVs23-26 or black lipid membranes27. They can directly be applied to the chip surface, where bilayer formation is taking place via a self-assembly process. The glass-bottom design of the nanoporous chip (Fig. 1) allows for air microscopy, which permits the straightforward automation of the system. In combination with a motorized stage multiple chips can be measured at the same time, with each field of view containing thousands of sealed cavities for analysis.
<img alt="Figure 1" src="/files/ftp_upload/53373/53373fig1.jpg" /> 
Figure 1. Design of multiplexed nanopore biochips. A) A silicon-on-insulator (SOI) wafer is structured by reactive-ion etching. Approximately 1,150 individual chips are fabricated from each wafer with identical properties and quality. B) Each chip comprises 250,000 individual microcavities with nano apertures. Scale bar: 200 &#181;m. C) Each cavity is addressable via multi-spectral fluorescence read-out. An intransparent top layer blocks the fluorescent signals from the buffer reservoir, making the biochip compatible with inverted fluorescence microscopes. D) Atomic force microscopy (AFM) imaging reveals evenly arranged pore openings and surface roughness of the silicon dioxide layer of 3.6 nm (n = 40) optimal for vesicle fusion. Scale bar: 5 &#181;m. E) Scanning electron microscopy (SEM) image shows a cross-section through the nanopore allowing access to the femtoliter cavities inside the silicon chip. This figure was reused with permission from 21. <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
All data analysis is performed using freeware to guarantee unrestricted access for end users. Time series are analyzed using free image processing software and a custom build curve analysis software enabling batch processing and straightforward correlation of large datasets with multiple fluorescent channels and thousands of curves.
The model protein used in this protocol is the mechanosensitive channel of large conductance (MscL) channel protein derived from E. coli. It functions as a valve to release osmotic shock in nature, but was modified in such a way that rationally designed synthetic functionalities can covalently be attached to the channels constriction side. Via charge-repulsion of the covalently bound activator (MTSET) the channel is triggered to open, creating a nano-valve. Small molecules like ions, water, small proteins, but also small fluorophores can permeate through the channel. Here, the protein is used as a model to demonstrate the ability of the system to detect protein-mediated translocation.

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			<td height="21" style="height:21px;width:389px;">Reagent</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">[2-(Trimethylammonium)ethyl] 
			methanethiosulfonate</td>
			<td style="width:232px;">Toronto Research Chemicals Inc.</td>
			<td style="width:305px;">T792900</td>
			<td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &#176;C. Use immediately (30 minutes) after solubilization in buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">1 ml gas-tight syringe</td>
			<td style="width:232px;">Hamilton</td>
			<td style="width:305px;">#1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">10 ml round flask</td>
			<td style="width:232px;">Schott Duran</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2.7 mm glas beads</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">N032.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2-Propanole</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">9866.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">30 cm Luer-Lock Extension Tube</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Acetone</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5025.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Bio-Beads SM-2 Adsorbent</td>
			<td style="width:232px;">Bio-Rad</td>
			<td style="width:305px;">152-3920</td>
			<td>need to be activated before first use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:389px;">CaCl2 Dihydrat</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">HN04.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Calcein</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:305px;">C0875</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Chloroform reagent grade</td>
			<td style="width:232px;">VWR Chemicals</td>
			<td style="width:305px;">22711324</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:389px;">DOPEATTO390</td>
			<td style="width:232px;">ATTO-TEC</td>
			<td style="width:305px;">AD 390-165</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Ethanol absolute</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">32205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt Single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">460 60 51V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt-F single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">91 66 017V</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:389px;">Keck clips</td>
			<td style="width:232px;">Schott</td>
			<td style="width:305px;">KC29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">L-&#945;-Phosphatidylcholine, 20% (Soy)</td>
			<td style="width:232px;">Avanti Polar Lipids</td>
			<td style="width:305px;">5416016</td>
			<td>store under inert gas at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NaCl 99.5% p.a.</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">3957.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanopore E100 wafer/chips</td>
			<td style="width:232px;">Micromotive (Mainz/Germany)</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nucleopore Track-Etch Membrane 0.4 &#181;m</td>
			<td style="width:232px;">Whatman</td>
			<td style="width:305px;">800282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oregon Green Dextran 488 (70 kDa)</td>
			<td style="width:232px;">life Technologies</td>
			<td style="width:305px;">D-7173</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oy647</td>
			<td style="width:232px;">Luminartis (M&#252;nster/Germany)</td>
			<td style="width:305px;">OY-647-T-1mg</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Rotilabo-syringe filtration, unsterile, pore-size 0.22 &#181;m</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">P 818.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sephadex G-50</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">G5080</td>
			<td>column material for size exclusion chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Silastic MDX4-4210</td>
			<td style="width:232px;">Dow Corning</td>
			<td style="width:305px;"></td>
			<td>curing agent for chip fixation onto cover glass support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">sticky-Slide 8-well</td>
			<td style="width:232px;">ibidi</td>
			<td style="width:305px;">80828</td>
			<td>multi-well chamber for the mounting onto glass slides (chip holder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Three-way stopcock blue</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744410001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Tris Pufferan 99.9% Ultra Quality</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5429.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Triton-X 100</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">6683.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Whatman 0.2 &#181;m cellulose nitrate membrane filter</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">NH69.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Equipment</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi 461 water bath</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi Rotavapor RE 111</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Cary Eclipse Fluorescence Spectrophtometer&#160;</td>
			<td style="width:232px;">Varian</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">LiposoFast Mini Extruder</td>
			<td style="width:232px;">Avestin</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Membrane pump&#160;</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">15430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanosight Nanoparticle Tracking Microscope</td>
			<td style="width:232px;">Malvern / Nanosight</td>
			<td style="width:305px;">LM 14C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NyONE microscope</td>
			<td style="width:232px;">Synentec</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Pump control</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">CVC 2II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sonicator bath Sonorex RK100H</td>
			<td style="width:232px;">Brandelin electronic</td>
			<td style="width:305px;">31200001107477</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Vaccum pump RC5</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">1805400204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Water bath W13</td>
			<td style="width:232px;">Haake</td>
			<td style="width:305px;">002-9910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Plasma Cleaner PDC-37G</td>
			<td style="width:232px;">Harrick Plasma</td>
			<td style="width:305px;">PDC-37G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Software</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">ImageJ</td>
			<td style="width:232px;">Open Source</td>
			<td style="width:305px;">http://imagej.nih.gov/ij/</td>
			<td>scientific image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NanoCalcFX</td>
			<td style="width:232px;">Freeware</td>
			<td style="width:305px;">http://sourceforge.net/projects/nanocalc/</td>
			<td>data analysis/evaluation software for massive transport kinetic datasets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Analytical Software</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>data acquisition and analysis software for nanoparticle tracking microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Temperature Comms</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>temperature controle software for nanoparticle tracking microscope</td>
		</tr>
	</tbody>
</table>

membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

Pore-spanning membranes can easily be created on the nanostructured chip surface in a self-assembled manner. However the underlying process is still delicate and influenced by many parameters like liposome size, monodispersity of the liposome population, lamellarity, lipid- and salt-concentration and chemical surface properties. Most of these parameters have been carefully characterized and standardized in the above protocol. Other parameters however should be checked during every new pre...

Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

The overall goal of this nano-structure chip-based platform is to analyze membrane protein function on the single molecular level in a highly parallel manner. Through its ability to resolve single proteins, new insights into membrane transport and flux are achieved. This method can help answer key questions in the membrane protein field, such as the conductivity or transport behavior of single channels or transporters, specially such with non-electrogenic cargo.The main advantage of this technique is its high parallelism derived from nano-structure chip design, featuring hundreds of thousand of nanopores and the fluorescent read out of transport events. The implications of this technique extend to what's creating best assays of effective libraries for membrane proteins. Because the chip-based platform enables highly parallel and automatic transport recordance.Generally, individuals new to this method will quickly learn how to handle the presented technique. The focus was always to create a user-friendly assay so that everyone interested can adopt it. This membrane transport assay utilizes a silicon on insulated wafer, engineered using reaction ion etching.About 1, 000 assay chips are fabricated from each wafer. Each chip contains 250, 000 microcavities. These cavities get sealed by lipid bilayers containing the transport proteins of interest.The membrane constitutes the sensor interface. Initially, fluorescent materials are contained to one side of the lipid bilayer, corresponding to the interior of the liposomes from which they are composed. Each cavity is addressable via a multispectral fluorescence readout.An opaque top layer blocks the fluorescent signals from the buffer reservoir, making the bio-chip compatible with inverted fluorescence microscopes. Using atomic force microscopy, the evenly arranged pore openings and surface roughness of the silicon-dioxide layer was measured at 3.6 nanometers, optimal for vesicle fusion. Scanning Electron Microscopy shows a cross-section through a nanopore, which accesses the femtoliter cavities inside the silicon chip.This video shows the preparation of the proteoliposomes, the preparation of the chips for the assay, and the basic assay execution. Connect an ethanol-washed round-bottom flask to a rotary evaporator. Dry the lipid film for 40 minutes at 300 millibar, 30 degrees Celsius water bath temperature, and maximal rotation speed to form a homogeneous lipid film on the glass wall of the flask.Then, using a high-vacuum pump, dry the lipid film for an additional 30 minutes at room temperature. Next, add five milliliters of buffer and eight ethanol-washed glass beads to the flask to make the multilamellar vesicles. Connect the flask to the rotary evaporator and rotate without vacuum at 50 degrees Celsius at maximum speed.After 45 minutes, the solution will appear milky, with no residual lipid film on the walls. Transfer the flask under an inert gas to an ultrasonic bath, and sonicate it for four minutes at room temperature. Sonication disrupts the liposomes making them unilamellar.Now, split the LUV solution into one milliliter aliquots, and perform five freeze thaw cycles using liquid nitrogen and a 40 degrees Celsius water bath. This leads to rupturing, rearrangement, and growth of the liposomes. At the last freezing step, store all samples at minus 80 degrees Celsius.Before reconstituting an LUV aliquot, extrude the liposomes by passing the aliquot through a 400 nanometer polycarbonate filter membrane 17 times, using a mini-extruder. Then, destabilize the LUVs by adding 4%Triton X-100 for 0.25%in solution. Incubate the mixture for five minutes at 50 degrees Celsius.Now, mix one part purified, modified MscL into 20 parts of the detergent-saturated liposomes, and continue the incubation for 30 minutes. After the incubation, take a 100 microliter aliquot, and add one volume of 30 millimolar calcine. Incubate this mixture for five minutes at 50 degrees Celsius.Now, remove the detergent from the calcine-treated and untreated sample. Add wet bio-beads in Tris buffer to each, and incubate them overnight at four degrees Celsius on a rotating plate. Before the experiment, test a preparation using an activity assay.To begin, mix elastomer base into the curing agent, and fully desiccate the mixture so it is aghast. The adhesive must be used within 30 minutes when ready. Meanwhile, taking precautions, clean a cover glass slide using five milliliters of chloroform, applied from a glass syringe, and let the slide dry.Now, using a 10 microliter pipette, deposit the prepared adhesive onto the cover glass. Then, carefully remove a chip from the wafer board using fine tweezers. With the coated side of the chip up, deposit it onto the adhesive, and gently rub it into the slide to evenly distribute the adhesive.Do not let air bubbles get trapped below, or any adhesive get above onto the chip. Also, make the assembly so it fits into the eight-chamber slide holder. Now desiccate the assemblies for two hours at 65 degrees Celsius in a cabinet dryer.Later, remove the protective layer on the chips with a few solvent washes at 54 degrees Celsius. First place the mounted chips into a slide holder. Then, incubate them in three solvent baths for 10 minutes each.Isopropanol, acetone, and isopropanol. Next, carefully and thoroughly wash the chips with ultra pure water. Complete the process by drying the chips under a gentle stream of nitrogen gas.Now, prepare an eight-well sticky slide and put it backwards on the bench. Then, carefully lift and press the cover slides onto the sticky slide with the chips facing the wells. Let the assembly rest for a few minutes before proceeding.Before proceeding with the translocation assay, wash the chip assembly thoroughly, as described in the text protocol, finishing with a Tris buffer bath. Next, exchange the bath solution with DOPEd Tris buffer, containing five millimolar calcium chloride, and one micromolar Oy647 dye. Next, add proteoliposomes to the chip chamber for a final concentration of one milligram per milliliter, and wait one hour.After an hour, rinse the chips four times with 400 microliters of calcium-free Tris buffer. Now, add the controlled dye to each well to five micromolar, and proceed with the assay. Next, mount the chip holder to the epifluorescence microscope, and focus on the rim of the nanopore chip to find the microcavities.Once the microcavities are in focus, scan the nanopore array for a region that displays the highest ceiling ratio. Details are provided in the text protocol. Liposomes were prepared and checked for their size distribution and dispersion.The prepared liposomes were larger than the pore diameter for the assay, which is essential. Monodispersed liposomes are also desirable. Before the experiment, the modified MscL protein in the liposome's preparation was checked using a fluorescence dequenching assay.The ratio of liposomes harboring active modified MscL was adequate to proceed with the translocation experiment. On the biochip, solute translocation of a fluorescent target via the modified MscL channel was observed. The chemical modulator, MTSET, was used to open the engineered MscL channels via electrostatic repulsion.The subsequent decrease of fluorescence inside the cavity space was monitored. This process proved to be highly reproducible, with very consistent efflux events. The resulting efflux curves clustered in two distinct populations, corresponding to single channel, or multi channel, translocation events.The system was able to follow up to three uniquely fluorescing dyes with real-time analysis. This facilitates precise discrimination of positive results, mono-exponential efflux events, complex kinetics, lipid intrusions, and membrane ruptures. Once mastered, this technique can be executed in consecutive steps, namely chip preparation, sample preparation, imaging, and analysis, making the entire protocol modular.

membrane-transport-processes-analyzed-highly-parallel-nanopore-chip

Research

JoVE Journal

Biology

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...