Research reported in this publication is supported by the Air Force Office of Scientific Research Basic Initiative Grant FA9550-12-1-0464.

<ol>
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The authors have nothing to disclose.

Mechanosensation signifies one of the first sensory transduction pathways that evolved in living organisms. Using this phenomenon for studying and understanding the mechano-electrical properties of the DIB, is a crucial step toward functional stimuli-responsive materials. It involves the incorporation and activation of a mechanosensitive channel, MscL, in the DIB as a mechanoelectrical transducer and a strain gauge to detect tension increase in the lipid bilayer interface. On another note, the function of MS channels could be regulated through the basic material properties of lipid bilayers including thickness, intrinsic curvature, and compressibility. In light of the aforementioned, the micropipette-based technique provides a valuable tool allowing the researcher the ability to study MS channels in DIBs and provides insights into the structure of the lipid bilayer, as well as the lipid-protein interactions.
Over the past three decades, patch-clamp was the primary method to study MS channels, since it allows clamping of both voltage and tension. However, patch-clamp requires bulky equipment and not suitable for miniaturization, a property required for the engineering of sensory and conversion devices. DIBs due to their simplicity, stability, and compactness represent a suitable environment to study the activity of MscL. Here, we extend previous advances in the DIB formation techniques by proposing a micropipette-based technique, with the ability to control the size of droplets and bilayer interface, the chemical composition of each droplet, and the tension at the interface through dynamic stimulation. The technique consists of anchoring aqueous droplets, containing proteoliposomes, to the tips of coaxially opposing glass capillaries. The droplets are placed in a bath of organic solvent and when brought in contact a lipid bilayer forms at the interface.
The micropipettes are attached to piezoelectric oscillators, allowing horizontal displacement of the droplets. Dynamically compressing the droplets, results in an increase of interfacial tension at the water oil interface and therefore an increase in bilayer tension. Two major aspects differentiate this method from the similar and recently published contact bubble bilayer (CBB) technique37. Using the technique presented herein, the size of the bilayer is controlled using micromanipulators and thus the volumes of the droplets remain constant, unlike in the CBB method. In addition, the CBB technique calls for pressure pumps, which are not needed in the method presented in this paper making it simpler and easier to build.
We are able to incorporate and stimulate bacterial MscL for the first time without the use of a patch pipette or chemical modifications38. Since the system facilitates the formation of robust asymmetric lipid bilayer membranes, it more closely mimics the lipid asymmetry found in biological membranes. This allows us to study the effects of controlled membrane composition or asymmetry on the activity of MscL. Additionally, through image processing techniques, this method helps estimate the tension at the bilayer interface. This technique assists in understanding the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves the understanding of MscL response to membrane tension.
Although this method takes us a step closer toward a biomolecular stimuli-responsive material system and to a different physiological environment to study MscL, there are limitations to the system. Tension in this system cannot be clamped due to the presence of the lipid reservoir in the form of liposomes in each droplet, which tends to relieve tension at the oil/water interface. Therefore, at present mechanosensitive channels can be stimulated in DIBs only in a dynamic regime. The presence of air bubbles in the system significantly affects the precision and reproducibility of the experiments. Air bubbles present in the hydrogels could result loss if electrical connection.
While we describe the use of the micro-pipette based method for the stimulation of MscL, the technique could be used to study other types of MS channels and has the potential to be used by researchers to study a variety of biomolecules. For instance, similar setup has been used in our lab to study the mechanoelectrical response of a channel-free droplet interface bilayer membrane. Various proteins could be reconstituted and activated using this highly controlled setup, taking in consideration that the reconstitution environments of each biomolecule vary. The method described in this article touches on a considerably wider application potential that is only limited to the imagination of the researcher.

In the past decade, the assembly of artificial lipid bilayers has been substantially advanced through the development of the droplet interface bilayer method. Known as stable and robust, DIBs imposed themselves as alternative model systems to the classical painted (Mueller) and folded (Montal-Mueller) planar bilayers1. Although the idea of using droplets to create lipid bilayers dates back to the 1960s2, it has not gained popularity until recently. The first successful attempt was reported by the Takeushi group3, followed by several studies demonstrating bilayer formation using a network of droplets by the Bayley group4-6. More recently, encapsulation techniques were proposed by the Leo group7-9, who pioneered the concept of using DIBs as building blocks of novel stimuli-responsive material systems10. In previous studies, DIBs have proved their ability to respond to electrical9,11, chemical10,12, and optical stimuli13. Various biomolecules with different stimuli-responsive functionalities have been effectively stimulated when reconstituted in the DIB10,14. In light of these successful attempts an important question is raised: could the DIB respond to mechanical stimulus when appropriate biomolecules are incorporated? The interfacial forces acting on a DIB differ from those in other bilayer system15,16. Therefore, the tension in the bilayer held by the droplets could be controlled by regulating tension at the water-lipid-oil interfaces; a concept not applicable with the painted or folded bilayer systems.
MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased membrane tension17,18. In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell19 can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis20. Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique21-23. Reliable structural models explaining MscL&#39;s gating mechanism24,25 are proposed based on its homolog&#39;s crystal structure26,27, modeling28, and results of extensive experimentation 24,29-31. Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming a ~28 &#197; water-filled conductive pore21,24,32. It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel33. Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered22. This property of MscL made possible the design of various controllable valves34, primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs.
In this article, we present an original micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels under mechanical stimulation. We report for the first time, the response of DIBs to mechanical stimulus and the functional reconstitution of the V23T low-threshold mutant of MscL in DIBs35.
The experimental system consists of lipid encased aqueous droplets anchored to the tips of two opposing borosilicate glass micropipettes. When droplets are brought into contact a lipid bilayer interface is formed. This technique offers control over the chemical composition and size of each droplet (bulk), as well as the dimensions of the bilayer interface. In addition, asymmetric membranes with various lipid compositions in each leaflet could be easily formed. Having one of the micropipettes attached to a harmonic piezoelectric actuator, provides the ability to apply a pre-programmed single-cycle or oscillatory stimulus. Tension is delivered to the artificial membrane through the compression of both droplets supporting it. As a result of droplet deformation, the areas of water-lipid-oil interfaces increase, and simultaneously the angle between the droplets decreases, causing an increase in membrane tension and transient MscL activation. Through analysis of the shapes of the droplets during deformation, the tension created at the interface could be estimated. Even though the focus in this article is on the mechano-transduction properties of the DIB, we also emphasize that other types of biomolecules, such as alamethicin, can be activated by this multi-functional platform. We present here, all the technical aspects of preparing, assembling, and taking measurements with this new method in a step-by-step manner.

<table><tbody><tr><td>0.22 &amp;micro;m filter</td><td>Corning</td><td>430624</td><td></td></tr><tr><td>1,2-diphytanoy-sn-glycero-3-phosphocholine (DPhPC)</td><td>Avanti Polar Lipids</td><td>850356P</td><td>Purchased as lyophilized powder</td></tr><tr><td>34-gauge microfil</td><td>World Precision Instruments</td><td>MF24G-5</td><td></td></tr><tr><td>400 mL Centrifuge bottels</td><td>ThermoFisher</td><td>3141</td><td>Nalgene</td></tr><tr><td>Agilent Function/Arbitrary Waveform Generator, 20 MHz</td><td>Keysight Technologies</td><td>33220A</td><td></td></tr><tr><td>Ampicillian</td><td>ThermoFisher</td><td>BP1760</td><td>ACS Grade</td></tr><tr><td>Avanti&amp;reg; Mini-Extruder</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>Axio Scope.A1</td><td>Carl Zeiss</td><td>-</td><td></td></tr><tr><td>AxioCam HSm</td><td>Carl Zeiss</td><td>-</td><td></td></tr><tr><td>Axopatch 200B Amplifier&amp;nbsp;</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Pierce&amp;nbsp;</td><td>23225</td><td></td></tr><tr><td>BK Precision 4017B 10 MHz DDs Sweep/Function Generator</td><td>Digi-Key</td><td>BK4017B-ND</td><td></td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Dialysis tubing</td><td>7 Spectra/Por</td><td>132113</td><td>MWCO 8000, 7.5 mm diameter</td></tr><tr><td>DigiData 1440A system</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>DPhPC</td><td>Avanti</td><td>850356C</td><td></td></tr><tr><td>E-625 PZT Servo-Controller&amp;nbsp;</td><td>Physik Instrumente</td><td>E-526</td><td></td></tr><tr><td>FPLC System</td><td>Pharmacia Biotech</td><td>-</td><td></td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-33</td><td></td></tr><tr><td>Hexadecane, 99%</td><td>Sigma-Aldrich</td><td>544-76-3</td><td></td></tr><tr><td>Homoginizer</td><td>Wheaton</td><td>357426</td><td>15 mL</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>IPTG</td><td>Affymetrix</td><td>17886</td><td></td></tr><tr><td>IRGACURE&amp;reg; 2959</td><td>IRGACURE&amp;reg;</td><td>555047962</td><td></td></tr><tr><td>Isopore Membrane Filters&amp;nbsp;</td><td>EMD Millipore</td><td>VCTP02500</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>VWR International</td><td>BDH1133-4LP</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td>ACS Grade</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>7100</td><td>ACS Grade</td></tr><tr><td>Kimble-Chase</td><td>Kontes</td><td>420401-1515</td><td>Flex-Column</td></tr><tr><td>LED-100 UV Spot Curing System</td><td>Electro-Lite, corp.</td><td>81170</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>Manual Patch-Clamp Micromanipulators</td><td>Thorlabs</td><td>PCS-520N</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ThermoFisher</td><td>M33</td><td>ACS Grade</td></tr><tr><td>Microelectrode Holder&amp;nbsp;</td><td>World Precision Instruments</td><td>MEH1S</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>MOPS, minimum 99.5% titration</td><td>Sigma-Aldrich</td><td>M1254-100G</td><td></td></tr><tr><td>N&lt;sub&gt;2 &lt;/sub&gt;Gas</td><td>Airgas</td><td>UN1066</td><td></td></tr><tr><td>NaCl</td><td>EMD</td><td>SX0420-1</td><td>ACS Grade</td></tr><tr><td>Ni NTA agarose beads</td><td>Qiagen</td><td>1000632</td><td></td></tr><tr><td>Optically Clear Cast Acrylic Tube, 2-1/2&quot; OD x 2&quot; ID</td><td>McMaster-Carr</td><td>8486K545</td><td></td></tr><tr><td>P-601 PiezoMove Flexure-Guided Linear Actuator</td><td>Physik Instrumente</td><td>P-601</td><td></td></tr><tr><td>PAGE gel</td><td>Bio-Rad</td><td>456-9033</td><td></td></tr><tr><td>Parafilm M&amp;reg; All-Purpose Laboratory Film</td><td>Parafilm&amp;reg;</td><td>PM999</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Poly(ethylene glycol)1000 dimethacrylate</td><td>Polysciences, Inc.</td><td>15178-100</td><td></td></tr><tr><td>Polycarbonate (PCTE) Membrane Filters, Black, 0.4 Micron, 25mm, 100/Pk</td><td>Sterlitech Corporation</td><td>&lt;a href=&quot;http://www.sterlitech.com/filters/membrane-disc-filters/polycarbonate-membranes/hydrophilic-polycarbonate-membrane-filters/hydrophilic-polycarbonate-membrane-filter-pctb0425100.html&quot;&gt;PCTB0425100&lt;/a&gt;</td><td></td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P5405-500G</td><td></td></tr><tr><td>Powder Free Soft Nitrile Examination Gloves&amp;nbsp;</td><td>VWR International</td><td>CA89-38-272</td><td></td></tr><tr><td>Replacement Gasket 1.0mm</td><td>World Precision Instruments</td><td>GO1-100</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L5750</td><td></td></tr><tr><td>Silver wire</td><td>GoodFellow</td><td>147-346-94</td><td>Different diameters could be used depending on the application&amp;nbsp;</td></tr><tr><td>Sodium Azide</td><td>Affymetrix</td><td>21610</td><td></td></tr><tr><td>Test tubes</td><td>ThermoFisher</td><td>14-961-27</td><td>12 x 130 mm</td></tr><tr><td>Tryptone</td><td>ThermoFisher</td><td>BP1421</td><td></td></tr><tr><td>Ultracal 30K</td><td>Millipore</td><td>UFC803024</td><td>Amicore Ultra 30 MWCO</td></tr><tr><td>VWR Light-Duty Tissue Wipers</td><td>VWR International</td><td>82003-820</td><td></td></tr><tr><td>VWR Scientific 50D Ultrasonic Cleaner</td><td>VWR International</td><td>13089</td><td></td></tr><tr><td>Water Purifier</td><td>Barnstead</td><td>D11931</td><td></td></tr><tr><td>Yeast</td><td>ThermoFisher</td><td>BP1422</td><td></td></tr><tr><td>&amp;beta;-octylglucopyranoside</td><td>Anatrace</td><td>O311S</td><td></td></tr></tbody></table>

multifunctional, micropipette-based method for incorporation and stimulation of bacterial mechanosensitive ion channels in droplet interface bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Figures 1 and 2 display the experimental setup and equipment used to record protein activity in the course of mechanical stimulation of the lipid bilayer membrane. To minimize electrical noise into our measurements, the workstation is placed within a lab-made Faraday cage, grounded to a ground connection on the AxoPatch 200 B Amplifier.
Formation of a stable insulating lipid bilayer is a key step in this study. In this arrangement, a lipid monolayer assembles at the oil/water interface of the aqueous droplets immersed in a bath of an organic solvent. When droplets are placed in contact, excess oil is eliminated, and the opposing lipid monolayers thin to a two-molecule thick lipid bilayer. The most common technique used in bilayer characterization is voltage-clamp. With voltage-clamp, the voltage across the bilayer is maintained at a constant value while the current is measured. Figure 4 portrays a typical real-time current recording of the initial bilayer formation. Knowing the specific capacitance (~0.6 &#956;F/cm2)5 of the DPhPC lipid bilayer, the area of the formed bilayer could be calculated. The bilayer area could be controlled by changing the position of the droplets (Figure 4A). Using the piezoelectric actuator, different types of waveforms (sinusoidal, square, triangular, etc.) at different frequencies, amplitudes, and duty cycles could be applied to the droplets to horizontally and axially oscillate them and thus, bilayer tension and area could be altered (Figure 4B).
When the DIB is mechanically stimulated, while maintaining a constant DC potential across the membrane, a low-threshold (gain-of-function) V23T mutant of MscL generates reliable activities including mainly sub-conductive states and occasionally full opening events (Figure 5). These events are identical to those recorded using the patch-clamp technique from intact inner E. coli membranes and liposomes reconstituted with the purified V23T MscL. The results in Figure 5 prove that gating occurs in response to an increase in tension, since all current spikes are observed at peak compression. At peak compression, the relative areal expansion of the droplets is maximal and therefore, tension at the interface is maximal.
Alamethicin, a voltage-gated ion channel and one of the most studied peptides, increases the membrane permeability when a DC voltage is applied across the membrane36. The ability of the lipid bilayer interface to host transmembrane proteins and peptides is also tested by performing voltage-gating current recordings using alamethicin peptide. Alamethicin is mixed with the phospholipid solution to a final concentration of 100 ng/ml. Figure 6 shows the current measurements under voltage clamp (+115 mV). The droplets in this experiment are pulled apart in order to achieve small bilayer interface and thus higher resistance and smaller capacitance. The gating behavior of the Alamethicin peptide is shown through the discrete steps of current (Figure 6). The histogram on the right side of the plot shows the changes in conductance from the base level (0.0962 nS), which is basically the first conductance level of the channel itself.
<img alt="Figure 1" src="/files/ftp_upload/53362/53362fig1.jpg" /> 
Figure 1: A schematic describing the main parts and dimensions of the oil reservoir. The oil reservoir is manufactured at the machine shop at Virginia Tech. It consists of a machined cylindrical acrylic tube glued to the surface of an acrylic sheet. The dimensions and design can be modified to accommodate different applications or more than two micropipettes. <a href="https://www.jove.com/files/ftp_upload/53362/53362fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53362/53362fig2.jpg" /> 
Figure 2: Experimental setup and micropipettes preparation. (A) The standard workstation for forming, mechanically stimulating, and characterizing the interface bilayers includes a microscope, 3-axis manipulators, a digital camera, piezoelectric oscillator, vibration isolation table, and a Faraday cage (not shown). (B) The experimental setup consists of two opposing PEG-DMA hydrogel filled micropipettes horizontally positioned within a bath of Hexadecane oil. Each of the micropipettes contains an Ag/AgCl electrode to provide electrical connection. A third micropipette filled with proteoliposome solution is used to form the droplets at the tip of the other micropipettes. (C) The DIB current response could be measured using a combination of the patch amplifier and the low-noise data acquisition system. (D) A closed up picture showing the aqueous droplets formed at the tip of the micropipettes. (E) Ag/AgCl electrodes are made by dipping the tip of two 250 &#956;m silver wires in bleach. The electrodes are then fed through two borosilicate glass capillaries filled with PEG-DMA hydrogel, which is cured with UV light to solidify. A straight microelectrode holder with male connector is used to connect one of the micropipettes to the headstage of the patch amplifier.
<img alt="Figure 3" src="/files/ftp_upload/53362/53362fig3.jpg" /> 
Figure 3: Images illustrating the formation of droplet interface bilayers. (A) A 10 &#956;m micropipette filled with proteoliposomes is positioned under the microscope in proximity to the micropipette tips. Using a syringe connected to the micropipette, dispense small volumes of the proteoliposomes to form spherical droplets to desired volume. Let the monolayer form by allowing the droplets to sit for ten min. Bring the droplets into contact; the bilayer will form after all oil at the interface is eliminated. (B) While the bilayer is formed, the chemical composition at both sides of the interface could be controlled by injecting desired chemicals using a micro-sized micropipette. (C) The droplets at the moment of first contact. (D) The droplets when the lipid bilayer is formed.
<img alt="Figure 4" src="/files/ftp_upload/53362/53362fig4.jpg" /> 
Figure 4: Real-time measurements show both the initial thinning and subsequent expansion of the interface. (A) Current measured in the course of bilayer formation through the application of a triangular electrical potential. The magnitude of the measured current is directly proportional to the capacitance, and thus the area of the bilayer interface. The closer the droplets are brought together, the bigger the area of the interface and vice versa. (B) Upon application of mechanical excitation, the area of the bilayer interface increases and decreases at the same frequency as the stimulating signal.
<img alt="Figure 5" src="/files/ftp_upload/53362/53362fig5.jpg" /> 
Figure 5: Real-time measurements show the response of the bilayer to mechanical excitation as well as the gating of the V23T mutant of MscL. The shape of the current response is sinusoidal, which relates to a sinusoidal change in bilayer capacitance as a result of the bilayer area change. The current spikes, occurring at the peak of each cycle, indicate sub-conductance gating of the V23T mutant. A polar plot further indicates that gating occurs at peak compression, which reflects an increase in tension at the bilayer interface.
<img alt="Figure 6" src="/files/ftp_upload/53362/53362fig6.jpg" /> 
Figure 6: Current measurements under voltage clamp and corresponding histogram of conductance levels for gating activity of incorporated Alamethicin channels. The gating behavior of the Alamethicin peptide is shown through the discrete step-wise increase in current. The conductance levels match very well with previous measurements performed by our research group at Virginia Tech7.

Watch this Scientific Journal Video about Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers at JoVE.

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

multifunctional-micropipette-based-method-for-incorporation

Nanyang Technological University

Research

JoVE Journal

Biology

10.8K Views.  Virginia Polytechnic Institute and State University.  Bacteriële mechanosensitieve kanalen kunnen worden gebruikt als mechanoelectrical transducers in biomoleculaire inrichtingen. Droplet-interface dubbellagen (DIB), cel-geïnspireerde bouwstenen om dergelijke apparaten, vertegenwoordigen nieuwe platforms op te nemen en te stimuleren mechanosensitieve kanalen. Hier tonen we een nieuwe micropipet gebaseerde werkwijze voor het vormen van DIB, waardoor de studie van mechanosensitieve kanalen onder mechanische stimulatie. 

Video: Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Biochemistry

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

Neuroscience

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Single-Cell Mechanobiology Using Fluorescence Micropipette Aspiration

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels. The sophisticated setup incorporates a precision-engineered borosilicate glass micropipette connected to a finely regulated water reservoir and pneumatic aspiration system, facilitating controlled pressure application with increments as refined as &#177; 1 mmHg. A significant enhancement is the integration of epi-fluorescence imaging, allowing for the simultaneous observation and quantification of cell morphological changes and intracellular calcium fluxes during aspiration. The fMPA assay, through its synergistic combination of epi-fluorescence imaging with micropipette aspiration, sets a new standard for the study of cell mechanosensing within mechanically challenging environments. This multifaceted approach is adaptable to various experimental setups, providing critical insights into the single-cell mechanosensing mechanisms.

Author Spotlight: Advancing Live-Cell Mechanobiology Through Fluorescence Microaspiration

The exploration of cellular behavior under mechanical stress is pivotal for advances in cellular mechanics and mechanobiology. We introduce the Fluorescence Micropipette Aspiration (fMPA) technique, a novel method combining controlled mechanical stimulation with comprehensive analysis of intracellular signaling in single cells. This technique investigates new in-depth studies of live-cell mechanobiology.

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

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