Name: single-molecule imaging of lateral mobility and ion channel activity in lipid bilayers using total internal reflection fluorescence (tirf) microscopy
Uploaded: 2023-02-17T14:40:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy at JoVE.

We thank Beate Nitschke for her help with protein preparation and Robin Ghosh, Michael Schweikert (Stuttgart), and Maximilan Ulbrich (Freiburg) for insightful discussions. This work was supported by the Stuttgart Research Center Systems Biology (SRCSB) and a grant from the Baden W&#252;rttemberg Foundation (BiofMO-6 to SN).

We declare no conflicts of interest.

The protocol presented here provides an introduction to the use of DIB membranes to study the interplay between lateral ion channel movement and channel function using single-molecule TIRF microscopy. To obtain the best possible data, the preparation of stable DIB membranes with as many well-separated channels as possible is crucial for obtaining time series of individual particles, which can be analyzed satisfactorily.
Critical parameters to be optimized include the choice of lipid, the lipid concentration in the oil phase, and the protein and detergent concentrations in the aqueous droplets. The lipids employed are unusual, in that they show no clear phase transition at low temperatures. DPhPC is a commonly used lipid to produce stable membrane systems40. In principle, any lipid which maintains its fluid environment at low temperatures may be suitable for this application. In addition, the lipid should not be sensitive to oxidation. The detergent concentration in the droplets should be as low as possible to avoid membrane rupture. Stable membranes and good protein incorporation rates are generally achieved with detergent concentrations below the critical micelle concentration (cmc), given that the membrane protein does not precipitate.
If the DIB membranes do not tolerate specific detergents21,41, or if the proteins do not integrate from low detergent solution into the DIB membranes, the protein channels can first be reconstituted into small unilamellar lipid vesicles (SUVs), which are then fused to the DIB membranes from the droplet side, as has been successfully shown for E. coli MscL42. Sometimes, DIB membranes do not form because the lipid concentration in the oil phase is too low. To prevent DIB membranes from bursting, one should also be aware that the osmotic pressure between the hydrogel and the droplet must be precisely balanced without affecting the Ca2+-flux from cis to trans excessively. Optimized agarose thickness and mesh size seem to be crucial to observe the diffusion of membrane proteins. Any drying of the agarose layer should be avoided. The thickness can be determined using atomic force microscopy17. By varying the agarose concentration, volume, and rotation speed during spin coating, the mesh size and thickness of the hydrogel can be optimized. Note, however, that hydrogel layer thickness affects image contrast. To capture membrane proteins in DIBs, the agarose hydrogel can be replaced by custom-synthesized, non-crosslinked, Ni-NTA-modified, low-melting agarose to trap them via a His-tag17. An excessively high fluorescence background is often caused by rupture of the DIB membranes. This is particularly a problem with multi-well chambers, as the Ca2+-sensitive dye diffuses into the hydrogel. In this case, adjacent wells should be avoided. Fluorescence bleaching of the Ca2+-sensitive dye above the membrane should not be a significant limiting factor, as it is exchanged by unexcited dyes in the bulk of the droplet (Figure 3A) outside the TIRF evanescent field. The localization precision for the protein is given by the accuracy of fitting the spots and the pixel size.
Weak fluorescence signals can be caused by low Ca2+-flux through the channel. Possible reasons include: (i) inaccurate TIRF settings (e.g., laser intensity), (ii) the osmotic Ca2+ pressure across the membrane, or (iii) the intrinsic Ca2+-permeability of the channels is too low. To cope with the first issue, laser intensity, TIRF angle, and camera gain need to be optimized. The latter two issues can be overcome by the application of an electrical potential across the membrane2,43. However, the application of external voltages can distort the result, as electrical effects can influence the channel opening of ligand-gated or mechanosensitive ion channels that are actually not voltage-controlled. Examples of such channels are the mitochondrial protein translocase TOM-CC27, and its channel-forming subunit Tom4026,44,45,46. Finally, it should be noted that, inserting membrane proteins into DIB membranes in a specific orientation to achieve a desired functionality is tricky, and quantitative studies are rare47,48. In some cases, the orientation of the integrated proteins is random. This is a serious problem for studying membrane proteins, because certain membrane proteins are activated on only one side of the membrane.
TIRF microscopy is a powerful method for addressing single-molecule events in planar supported membranes49. Examples include assembly and folding pathway elucidation of channel proteins such as &#945;-hemolysin50, perfringolysin O51, and OmpG52. These studies included FRET as an additional technique. In addition, activation of the mechanosensitive ion channel MscL has previously been studied by mechanical stimulation of supported DIB bilayers42 using current measurements. Based on this work, future studies could combine the platform described here with single-molecule FRET experiments to address mechanosensitive channels at the single-molecule level in an optical manner17. Injection of buffer into the droplet, stretching the inner DIB monolayer, or targeted binding of individual channels to the underlying hydrogel can be used to further study not only the physical mechanism of mechanically activated channels, which respond to membrane tension and/or curvature as shown for MscL and MscS, the two-pore domain K+-channels, TREK-1, TREK-2, and TRAAK, and PIEZO (for review, see53), but also local binding to the cellular cytoskeleton, as shown for the touch-sensitive ion channel NOMPC54,55.

The present protocol aims to describe how to study the correlation between membrane mobility and ion channel permeability of membrane proteins in polymer-supported droplet-interface bilayer (DIB) membranes1,2,3.
The present technique complements an impressive array of advanced optical and surface analytical tools, such as single particle tracking4,5, fluorescence correlation spectroscopy6,7, and high-speed atomic force microscopy8,9,10. These provide valuable insights into the dynamic composition and structure of membranes that influence membrane-based reactions11,12,13. While the movement and lateral diffusion of proteins depends on the local density of proteins in the membrane, individual protein molecules can also be trapped in lipid rafts14 and by protein-protein interactions15,16. Depending on the protein domains protruding from the membrane into the extracellular environment or cytosol, protein mobility can vary from highly mobile to completely immobile. However, due to the complexity of the membrane and its peripheral structures, it is often difficult to decipher the interplay between the nature of lateral mobility and protein function17.
DIB membranes have proven to be an efficient platform for biophysical single-molecule analyses of membrane proteins18,19,20,21,22. They are formed by lipid self-assembly through the contact of aqueous droplets with hydrogel-supported substrates in a lipid/oil phase. Similar to the commonly used supported lipid bilayers (SLBs)1,23,24,25, DIBs allow local tuning of the lateral mobility by temporary or permanent binding of proteins to the polymer matrix when functionalized with suitable ligands17. The latter can serve as a model system for biochemical processes in cell membranes with a heterogeneous protein distribution10.
The experimental approach described here relies on the fluorescence of Ca2+-sensitive dyes to measure the Ca2+ ion flux through individual channels in close proximity to the membrane2,22 using TIRF microscopy. This optical approach limits the illumination of the sample to a distance close to the membrane, which, due to the physical properties of the evanescent excitation light, leads to a significant reduction of the fluorescence background. The latter is a prerequisite if high spatial and temporal resolution is required for the detection of single molecules. In contrast to classical electrophysiological methods26,27, no membrane voltages are required to study ion flux through individual channels. Furthermore, the method does not require labeling with fluorescent dyes or molecules that could interfere with the lateral movement of the channels in the membrane.
The method is particularly useful for studying protein channels embedded in the membrane at the single molecule level without using classical electrophysiology. Using mitochondrial protein-conducting channel TOM-CC from Neurospora crassa28,29,30 and OmpF, which supports the diffusion of small hydrophilic molecules across the outer membrane of Escherichia coli17,31, we illustrate how the membrane mobilities and channel activities of the two proteins can be studied and correlated. We suggest that this approach, although optimized for TOM-CC and OmpF, can be readily applied to other protein channels.

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			<td>Diener Electronics</td>
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			<td>Resource Q 1 mL</td>
			<td>Cytiva</td>
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			<td>Silicon oil AR 20</td>
			<td>Sigma-Aldrich</td>
			<td>10836</td>
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			<td>Sodium dodecyl sulfate</td>
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			<td>Super LoLux camera</td>
			<td>JVC</td>
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			<td>Stereomicrosope&#160;</td>
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			<td>Thermoshaker</td>
			<td>Gerhardt</td>
			<td>THL 500/1</td>
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			<td>Ti-E Fluorescence microscope&#160;&#160;</td>
			<td>Nikon</td>
			<td>MEA53100</td>
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			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>9090.3</td>
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			<td>Tryptone</td>
			<td>Roth</td>
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			<td>Ultrasonic bath&#160;</td>
			<td>Bandelin Sonorex</td>
			<td>RK 100</td>
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			<td>Vaccum pump</td>
			<td>Vacuubrand</td>
			<td>MD 4C NT</td>
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		<tr>
			<td>White light source for epifluorescence illumination (100 W)</td>
			<td>Nikon</td>
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			<td>TIRF microscope&#160;</td>
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			<td>Yeast extract</td>
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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.

Real-time electrode-free optical single-channel recording reveals the interplay between lateral protein movement and the function of individual ion channels in DIB membranes. Reconstitution of the mitochondrial TOM core complex (Figure 1A) into a DIB membrane (Figure 4D) illustrates a strong temporal correlation between lateral mobility and ion permeability (Figure 5A). The TOM-CC gating appears to be sensitive to the mode of lateral movement17. Moving channels show Ca2+ flux through their pores and high fluorescence point intensities. Trapped, non-moving molecules show low and medium fluorescence intensities. For the general protein import pore of mitochondria TOM-CC, this single molecule approach revealed a strong temporal correlation between lateral mobility and ion permeability, suggesting that the TOM-CC channel has mechanosensitive properties17. A lateral stop of freely moving TOM-CC molecules is accompanied by a partial or complete closure of the TOM-CC channel. Imaging DIB membranes with OmpF (Figure 1E and Figure 4F), which is almost fully embedded in the membrane, shows no stop-and-go effects (Figure 5B). Random stops of OmpF are not associated with a change in intensity and, thus, with the closing of its pores. Based on the fluorescence signals38, the positional accuracy of the individual channels can be estimated in the range between 5 and 10 nm. However, it should be noted that this accuracy cannot be achieved if the channels wobble slightly due to mobile anchoring with the agarose hydrogel, as shown, for example, for the TOM-CC molecules in an intermediate state with a root mean displacement of 120 nm (Figure 5A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64970/64970fig01.jpg" /> 
Figure 1: Isolation of TOM-CC. (A) Cryo EM structure of N. crassa TOM-CC30,39. Mitochondria from a N. crassa strain containing a Tom22 with a 6xHis tag were solubilized in DDM and subjected to Ni-NTA affinity chromatography (B) and anion-exchange chromatography (C). (D) SDS-PAGE of isolated TOM-CC. (E) Crystal structure (PDB, 1OPF) and (F) SDS-PAGE of purified E. coli OmpF. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64970/64970fig02.jpg" /> 
Figure 2: Flow chart of PMMA chamber assembly. Step 1: A glass coverslip is spin-coated with an agarose hydrogel. Step 2: The spin-coated coverslip is mounted in a custom-made PMMA microscopy chamber. Step 3: Additional low melt agarose is added into the inlet port of the PMMA chamber on a 35 &#176;C hot plate. Step 4: Lipid monolayers are formed around aqueous droplets at a buffer/oil interface (left) and on the agarose hydrogel/oil interface (right). Step 5: Individual aqueous droplets are pipetted into the wells of the PMMA chamber to form a lipid bilayer upon contact of the two lipid monolayers. Step 6: Formation of the DIB membranes is validated by Hofmann modulation contrast microscopy. Step 7: Images of selected areas of the DIB membranes with inserted ion channels are acquired by TIRF microscopy. Green: 0.75% agarose; yellow: 2.5% agarose containing Ca2+ ions; magenta: lipid/oil phase; dark blue: aqueous droplet buffer containing Ca2+-sensitive dye and protein. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64970/64970fig03.jpg" /> 
Figure 3: Experimental setup. (A) Schematic representation of a DIB membrane in a PMMA well. The bilayer rests on an ultrathin 0.75% agarose film to allow TIRF imaging of Ca2+-flux through an ion channel over time using a Ca2+-sensitive fluorescence dye (Fluo-8) in trans. (B) The Ca2+-flux is controlled exclusively by the osmotic pressure from cis to trans. This allows both the determination of the position in the membrane and the open-closed state of the channel. The channel shown here is the protein-conducting channel TOM-CC of N. crassa mitochondria30. (C) Trajectory of a single TOM-CC channel. Green: moving channel; yellow: non-moving channel. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64970/64970fig04.jpg" /> 
Figure 4: Imaging TOM-CC and OmpF in DIB membranes. (A) DIB membrane imaged by Hoffmann modulation contrast microscopy showing the bilayer contact area between the hydrogel and the droplet. (B) Broken DIB membrane imaged as in (A). Arrow, edge of PMMA chamber. (C) DIB membrane imaged by TIRF microscopy without protein channels. (D) DIB membrane with reconstituted TOM-CC, imaged by TIRF microscopy. The white squares mark spots of high (SH), intermediate (SI), and low (SL) intensity. (E) Fitting the fluorescence intensity profile of the three spots marked in (A) to two-dimensional Gaussian functions reveals the position of individual TOM-CCs and the Ca2+ flux through the channel. (F) DIB membrane with reconstituted OmpF. (G) Gaussian fit of the fluorescent spot marked in (F). In contrast to the two-pore &#946;-barrel protein complex TOM-CC, the three-pore &#946;-barrel OmpF reveals only one permeability state. Pixel size, 0.16 &#956;m.&#160;<a href="https://www.jove.com/files/ftp_upload/64970/64970fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64970/64970fig05.jpg" /> 
Figure 5: Channel activity correlates with lateral mobility of TOM-CC. (A) The fluorescent amplitude trace (top) and corresponding trajectory (bottom) of TOM-CC indicate that the open-closed channel activity of TOM-CC correlates with the lateral membrane mobility of the complex. The trajectory displays three permeability states. Green: fully open state; yellow: intermediate permeability state; red: closed channel state; red star: TOM-CC in the intermediate state wobbles around its mean position by about &#177;60 nm. The positional accuracies37 based on the fluorescence signals in the fully open and intermediate states range between 5 nm and 10 nm. (B) Fluorescent amplitude trace (top) and corresponding trajectory (bottom) of OmpF. OmpF reveals only one intensity level, regardless of whether it is in motion or trapped. The trajectory segments corresponding to the time periods of trapped molecules are marked in grey. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer&#160;</td>
			<td colspan="3">Reagent concentrations</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>A1*</td>
			<td colspan="3">20 mM Tris-HCl pH 8.5, 0.1 % (w/v) n-dodecyl-&#946;-D-maltoside (DDM), 10% (v/v) glycerol, 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>A2*</td>
			<td colspan="3">20 mM Tris-HCl pH 8.5, 0.1% (w/v) DDM, 10% (v/v) glycerol, 1 M Imidazole and 1 mM PMSF</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>B1*</td>
			<td colspan="3">20&#8201;mM HEPES pH 7.2, 0.1% (w/v) DDM, 2% (v/v) dimethyl sulfoxide (DMSO)</td>
			<td>100 mL&#160;</td>
		</tr>
		<tr>
			<td>B2*</td>
			<td colspan="3">20&#8201;mM HEPES pH 7.2, 0.1% (w/v) DDM, 1 M KCl, 2% (v/v) dimethyl sulfoxide (DMSO)</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td colspan="5">* Degas and pass through a 0.22 &#956;m filter before use.</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer solutions for TOM-CC isolation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer&#160;</td>
			<td colspan="3">Reagent concentrations</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>LB*</td>
			<td colspan="3">1% (w/v) tryptone, 1% (w/v) NaCl and 0.5% (w/v) yeast extract&#160;</td>
			<td>1100 mL</td>
		</tr>
		<tr>
			<td>C1</td>
			<td colspan="3">2 mM MgCl2, and ~750 units DNAse and 50 mM Tris-HCl pH 7.5&#160;&#160;</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>C2</td>
			<td colspan="3">50 mM Tris-HCl, pH 7.5</td>
			<td>50 mL&#160;</td>
		</tr>
		<tr>
			<td>C3</td>
			<td colspan="3">4% (w/v) sodium dodecyl sulfate (SDS), 2 mM &#946;-mercaptoethanol and 50 mM Tris-HCl pH 7.5</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>C4</td>
			<td colspan="3">2% (w/v) SDS, 500 mM NaCl and 50 mM Tris-HCl pH 7.5</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>C5</td>
			<td colspan="3">0.5% (w/v) octyl polyoxyethylene (octyl POE), 1 mM EDTA and 20 mM Tris pH 8.5&#160;</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td colspan="5">* Sterilize before use.</td>
		</tr>
	</tbody>
</table>
Table 2: Buffer solutions for OmpF isolation.

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