This work was supported by the Australian Research Council (ARC) Discovery Program (DP150101065) and the ARC Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL) (IH150100028).

1.&#160;tBLMs electrodes preparation
<ol>
	<li>Preparation of first monolayer coating
	<ol>
		<li>Immerse a freshly sputtered gold patterned electrode microscope slide in an ethanolic solution comprised of a 3 mM 1:9 ratio of benzyl-disulfide-tetra-ethyleneglycol-OH &#34;spacer&#34; molecules (benzyl disulfide comprised a four oxygen-ethylene glycol spacer, terminated with an OH group) and benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl &#34;tethered&#34; molecules. This creates the first layer coating to which a bilayer can be anchored. 
		&#8203;NOTE: The gold electrode is made by evaporating 100 nm, 99.9995% gold (5n5 gold) film onto custom 25 mm x 75 mm polycarbonate slides20.</li>
		<li>Incubate electrodes with the first layer at room temperature for at least 1 h.</li>
		<li>Rinse the gold electrodes by immersing in copious amounts of pure ethanol over 30 s.</li>
		<li>Use the gold electrode slide with the first monolayer directly for the next step or store in a jar full of pure ethanol.</li>
		<li>NOTE: To ensure the integrity of the first layer, minimize any direct contact to the gold portions of the slide</li>
	</ol>
	</li>
	<li>Assembling the first monolayer coated slide
	<ol>
		<li>Carefully take off one coplanar gold electrode slide from its container using tweezers, being sure not to make contact with the patterned areas where the tBLMs will form. 
		NOTE: Be mindful to identify the side of the slide onto which the gold is deposited.</li>
		<li>Air dry slide for 1 - 2 min in to remove any residual ethanol.</li>
		<li>Place gold electrode over a dry surface, ensure the gold electrode is correctly oriented with patterned gold surface facing up.</li>
		<li>Peel the transparent adhesive layer cover from a thin laminate and place over the 6 channels to define each well.</li>
		<li>Use a pressure roller to release any air between the slide and transparent adhesive layer, as shown in&#160;Figure 1A. 
		NOTE: The time required for this step will need to be optimized by the researcher. In this protocol, times are ranging from 2-3 min.</li>
		<li>Introduce as soon as practicable (within 1-2 minutes) the second lipid bilayer to the assembled first monolayer coated electrode for self-assembly to avoid damaging the first layer.</li>
	</ol>
	</li>
	<li>Preparation of second lipid bilayer
	<ol>
		<li>Add 6 &#181;L of 3 mM lipids of interest to the first well of the six wells slide. Do not let the edge of the micropipette tip touch the gold surface, which can damage the tethered chemistries on the electrode. 
		NOTE: The lipid mixture used in this work consisted of 3 mM 70% zwitterionic C20 diphytanyl-ether-glycero-phosphatidylcholine (DPEPC) and 30% C20 diphytanyldiglyceride ether lipids (GDPE) mixed with 3 mM cholesterol-PEG-Biotin in 50:1 molar ratio.</li>
		<li>Introduce 6 &#181;L of the lipid mixture to the other wells with a 10 s gap between each addition.</li>
		<li>Incubate each well for exactly 2 min at room temperature before exchanging the lipid mixture over the electrodes with a buffer such as PBS. Space the times for the addition and buffer exchange 10 s apart so each well is incubated with the lipid for exactly 2 min each.</li>
		<li>Wash 3 more times with 50 &#181;L of PBS buffer (pH 7.0). Be sure to leave 50 &#181;L of buffer over the electrodes at all times. Do not allow the electrodes to dry. 
		NOTE: Displacing the ethanol solvent with the aqueous solution in this way (the&#160;solvent exchange method) enables the rapid formation of a single lipid bilayer anchored to the gold electrode via the tethered chemistries.</li>
	</ol>
	</li>
	<li>Testing tBLM formation using electrical impedance spectroscopy (EIS) measurements
	<ol>
		<li>Insert prepared electrode slide into an AC impedance spectrometer (e.g., Tethapod). Ensure that the spectrometer is connected via a USB port to a computer running the software.</li>
		<li>Open the software, click&#160;Setup&#160;and open&#160;Hardware.</li>
		<li>Set the hardware settings to use 25 mV peak-to-peak AC excitation.</li>
		<li>Set frequencies between 0.1 and 10,000 Hz with two steps per decade for rapid impedance measures press ok.</li>
		<li>Click the&#160;Setup&#160;menu and open&#160;Model.</li>
		<li>Use an equivalent circuit model that describes the tethering gold electrode as a constant phase element in series with a resistor describing the electrolyte buffer and a parallel resistor-capacitor network to describe the lipid bilayer, and press&#160;OK.</li>
		<li>Press the&#160;Start&#160;button in order to start a real-time measurement of membrane capacitance (Cm) and membrane conduction (Gm). Cm&#160;values of typical tBLMs should be in the range of 12.5 nF to 15.5 nF for 10% tethered chemistries21,22.</li>
		<li>After running the protocol and finishing the experiment, save the data.</li>
		<li>Repeat the measurement with the next well.</li>
	</ol>
	</li>
</ol>
2.&#160;Laser irradiation
<ol>
	<li>Experimental setup 
	NOTE: The custom-made system is set up for each tBLM well individually.
	<ol>
		<li>Perform experiments in a light-proof box to minimize laser hazardously.</li>
		<li>Use an optics table to set up the experiment to reduce unwanted vibrations.</li>
		<li>Place the impedance reader, where is the gold slide is connected, on an XYZ stage and elevate such that it sits in the path of the laser source.</li>
		<li>Use coarse-fine focusing microscopic gearing to control the height of the laser source to achieve the appropriate precision.</li>
		<li>Target the laser path along the longitudinal axis of the electrode slide. 
		CAUTION: Always wear suitable laser safety glasses and maintain good laser safety protocols.</li>
		<li>Allow the selected tuned laser to stabilize before starting the experiment. 
		NOTE: A schematic of the experimental setup is illustrated in&#160;Figure 2A.</li>
	</ol>
	</li>
	<li>Alignment of laser and gold electrodes 
	NOTE: Before beginning, always assess the laser power output using a power-meter to ensure only very low wattages are delivered to the tBLMs.
	<ol>
		<li>Adjust either the laser path or the angle of the electrode such that the laser passes through the liquid covering the electrode and is just visible, evenly, at the gold surface.</li>
		<li>Adjust the laser beam light position for each experiment by raising or lowering the laser beam source using the fine adjustment while observing changes in membrane conductance.</li>
		<li>Lock the knob to secure the position of the laser path when there are no conductance changes are observed. 
		NOTE: Increased membrane conductance values will be generated when the laser interacts with the underlying gold electrode. It is, therefore, important to adjust the laser path such that no such interactions are possible.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Prepare the laser beam light alignment (where there is no change in membrane conductance), as shown in&#160;Figure 2, position 3.</li>
		<li>Add GNPs of interest (functionalized or bare) to the PBS buffer in which the tBLMs are immersed while the laser is switched OFF.</li>
		<li>Mix the PBS buffer surrounding the tBLMs gently three times, being careful not to touch the electrode.</li>
		<li>Incubate for 5-10 min at room temperature.</li>
		<li>Turn the laser ON to irradiate sample, using the correct aligned laser beam light position as seen in&#160;Figure 2, position 3.</li>
		<li>Use the appropriate combination of GNPs size, shape and concentration with laser light wavelength. 
		NOTE: The laser beam of set wavelength should couple to the corresponding GNP plasmon resonance frequency.</li>
		<li>Record measured current continuously (real-time measurements).</li>
		<li>Perform steps 2.2.1 - 2.3.7, omitting GNP addition for the control experiments.</li>
	</ol>
	</li>
</ol>
3.&#160;Statistical data analysis and presentation
<ol>
	<li>Export the data into a spreadsheet.</li>
	<li>Extract the membrane conductance parameter versus time.</li>
	<li>Use the recorded data after setting a laser beam light with the right position and prior to GNPs introduction.</li>
	<li>Normalize data by dividing the measured membrane conductance over the baseline membrane conductance. 
	NOTE: This confirms that relative changes in membrane conduction values elicited by introduced irradiated GNPs.</li>
	<li>Present data as plots of time (x-axis) versus normalized membrane conduction (y-axis).</li>
</ol>
4.&#160;Predict the amount of localized heat generated in the tBLMs from irradiated nanoparticles (thermal predictive model)
<ol>
	<li>Solve the radiation transfer problem according to Dombrovsky23, in order to calculate absorbed radiation power in irradiated nanoparticle solutions.</li>
	<li>Calculate the heat generation by incorporating the heat source due to absorbed radiation into the energy equation. 
	NOTE: For a detailed explanation of the numerical analysis of heat generation in the tBLMs from irradiated nanoparticles and the nanomaterial-electrode interface, refer to&#160;19.</li>
</ol>

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The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Prof Bruce Cornell is Director - Science and Technology at Surgical Diagnostics SDx tethered membranes Pty. Ltd.

This protocol describes the use of tBLM model with a coplanar electrode substrate in conjunction with a horizontal laser alignment set up that enables the real-time electrical impedance recording in response to laser irradiation of gold nanoparticles. The method of EIS recording presented here constructs a minimal list of experiments necessary to provide recording of ion current changes across the membrane, which corresponds to the heat generated by the coupled laser and gold nanoparticle...

The hyperthermic performance of irradiated gold nanomaterials offers a new class of minimally invasive, selective, targeted treatment for infections and tumors1. The employment of nanoparticles that can be heated by a laser has been used to selectively destroy diseased cells as well as providing a means for selective drug delivery2,3. A consequence of the photothermolysis phenomena of heated plasmonic nanoparticles is damage to the cell membranes. The fluid lipid bilayer membrane is considered a particularly vulnerable site for cells undergoing such treatments because denaturation of intrinsic membrane proteins as well as membrane damage can also lead to cell death4, as many proteins are there to maintain the ionic potential gradient across cell membranes. While the ability to determine and monitor heat transfer at the nanoscale is of key interest to the study and application of irradiated GNPs1,5,6,7, assessment and understanding of the molecular interactions between GNPs and bio-membranes, as well as the direct consequences of the laser-induced heating phenomena of embedded GNPs in biological tissues, are yet to be fully elucidated8. Therefore, a thorough understanding of the hyperthermia process of irradiated GNPs remains a challenge. As such, the development of a nanomaterial-electrode interface that mimics the natural surroundings of cells could provide a means by which to undertake an in-depth investigation of the heat transfer characteristics of irradiated gold nanoparticles within biological systems.
The complexity of native cell membranes is one of the significant challenges in understanding the irradiated GNPs interactions in cells. There have been various artificial membrane platforms developed to provide close simple bio-mimetic versions of natural lipid membrane architecture and functionality, including, but not limited to, black lipid membranes9, supported planar bilayer membranes10, hybrid bilayer membranes11, polymer-cushioned lipid bilayer membranes12&#160;and tethered bilayer lipid membranes13. Each artificial lipid membrane model has distinct advantages and limitations with respect to mimicking the natural lipid membranes14.
This study describes the employment of lipid membrane-coated electrodes as a sensor for assessing gold nanoparticle and lipid membrane interactions, using the tBLM model. The tBLM based biosensor detection scheme provides inherent stability and sensitivity13&#160;as tethered membranes can self-repair, unlike other systems (such as membranes formed by patch-clamp or liposomes) in which only a small amount of membrane damage results in their collapse15,16,17,18. Further, because tBLMs are of mm2&#160;dimensions, the background impedance is orders of magnitude lower than patch-clamp recording techniques, which enables a recording of changes in basal membrane ionic flux due to nanoparticle interactions. As a result of this, the present protocol can contrast changes in membrane conductance by bound GNPs that are excited by lasers whose powers are as low as 135 nW/&#181;m2.
The system presented here provides a sensitive and reproducible method for determining precise laser parameters, particle size, particle coatings and composition needed to design and develop thermal therapies. This is critical for the refinement of emerging photothermal therapies, as well as offering valuable information for detailed mechanisms of heat transfer within biological systems. The presented protocol is based on previously published work19. An outline of the protocol is as follows: the first section defines the tBLM formation; the second section outlines how to construct the setup and align the excitation laser source; the final section illustrates how to extract information from the electrical impedance spectroscopy data.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
		</tr>
		<tr>
			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

The gold substrate upon which tBLMs can be created is shown in&#160;Figure 1. A schematic of the experimental setup is presented in&#160;Figure 2.
Coplanar gold electrodes, as shown in&#160;Figure 1A, are made from 25 mm x 75 mm x 1 mm polycarbonate base substrate with patterned gold arrays. A transparent adhesive layer defines the six individual measuring chambers. The coplanar gold electrode allows the ...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

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Research

JoVE Journal

Biology

2.6K Views.  University of Technology Sydney. This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Video: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...