We thank Cedric A. J. Hutter and Markus A. Seeger from the Institute of Medical Microbiology at the University of Zurich, and Gonzalo Cebrero from Biozentrum of the University of Basel for collaboration in the generation of synthetic nanobodies (sybodies). We thank Maria Barthmes and Andre Bazzone from NANION Technologies for technical assistance. This work was supported by the Swiss National Science Foundation (SNSF) (PP00P3_170607 and NANION Research Grant Initiative to C.P.).

<ol>
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The authors declare no competing financial interests.

The technique presented here classifies nanobodies with inhibitory and non-inhibitory properties targeting electrogenic transporters. Assessing the substrate transport is possible due to the detection of the movement of charges through the transporter embedded in the membrane of proteoliposomes. Some of the critical steps during the setup of an experiment are reconstitution of active protein in liposomes, preparation of stable monolayers on SSM chips, and recovering of initial conditions after the application of the wash protocol to remove bound nanobody molecules. Once the membrane protein is reconstituted at an appropriate lipid-to-protein ratio, a general SSM protocol can be established using the native substrate. It is crucial to perform control experiments using protein-free liposomes to reveal noise currents that would need to be subtracted from the currents measured using proteoliposomes. However, if noise currents are too large, we recommend trying protein reconstitution using different lipids, or screen for buffer conditions that minimize these deleterious signals. After successfully establishing conditions for an SSM assay, screening of nanobodies can be performed. A very useful option that may help in faster screening of nanobodies is to perform high-throughput assays using a single SSM sensor chip. This reduces the time of manipulation of chips and buffers and reduces the costs. However, because during this type of assay multiple nanobodies are applied sequentially, it is important to ensure that the applied nanobody can be washed away after the measurement. A stringent washing cycle may need to be implemented to unbind some nanobodies in case that reduced peak current amplitudes are detected in the absence of nanobody. We recommend using, as the starting point, the washing conditions described here. If increasing the washing volume or the number of cycles does not help, individual chips would need to be used to screen each nanobody separately. In all the cases examined here, the binding of nanobodies was reversible and a high-throughput protocol could be applied. In our experimental setup, we could not recover the full amplitude of the initial peak current after measurements with nanobodies (Figure 1A; Figure 2C,D). However, in most cases, the magnitude of the peak currents recovered ranged between 80 and 95% of the amplitude measured before applying nanobodies (Fig. 2C,D). We speculate that this could be a consequence of washing away a fraction of the proteoliposomes adhered to the chip, or due to slow kinetics of unbinding of some inhibitory nanobodies, or a combination of both. In either case, it was still possible to continue assaying further nanobodies as electrogenic transport was measurable. This is shown in Figure 2D, where we screened six nanobodies using a single chip preparation.
The high-throughput characteristic is one of the most significant advances of the method presented. In addition, in contrast to other approaches, this method allows for the selection of inhibitory nanobodies targeting electrogenic transporters for which labeled substrates are not available. A fast identification of inhibitory nanobodies can help to speed up the research aiming to identify novel applications of nanobodies as drugs. Their apparent advantages compared to similar therapies such as antibody treatments are numerous, starting with a smaller size which helps them propagate further into tissues or cells, to their low production costs and high stability.
SSM-based electrophysiology has been used in the past for the characterization of electrogenic transporters in membrane vesicles29,38. These types of experiments are advantageous as they do not depend on the protein purification and reconstitution protocols. We speculate that performing the selection of inhibitory nanobodies using membrane vesicles is feasible. This would help to reduce costs and avoid manipulations of purified proteins.
SSM-based electrophysiology is a strong technique to screen for nanobody inhibitors of multiple membrane proteins that exhibit electrogenic transport. We envision that SSM-based electrophysiology will become an important tool for the selection of inhibitory nanobodies and other antibodies with potential clinical applications.

Antibodies are composed of two identical heavy chains and two light chains that are responsible for the antigen binding. Camelids have heavy-chain only antibodies that exhibit similar affinity for their cognate antigen compared to conventional antibodies1,2. The single variable domain (VHH) of heavy-chain only antibodies retain the full antigen-binding potential and has been shown to be very stable1,2. These isolated VHH molecules or &#34;nanobodies&#34; have been implemented in studies related to membrane proteins biochemistry as tools for stabilizing conformations3,4, as inhibitors5,6, as stabilization agents7, and as gadgets for&#160;structure determination8,9,10. Nanobodies can be generated by the immunization of camelids for the pre-enrichment of B-cells that encode target-specific nanobodies and subsequent isolation of B cells, followed by cloning of the nanobody library and selection by phage display11,12,13. An alternate way to generate nanobodies is based on in vitro selection methods that rely on the construction of libraries and selection by phage display, ribosome display, or yeast display14,15,16,17,18,19,20. These in vitro methods require large library sizes but benefit from avoiding animal immunization and favor the selection of nanobodies targeting proteins with relatively low stability.
The small size of nanobodies, their high stability and solubility, strong antigen affinity, low immunogenicity, and relatively easy production, make them strong candidates for the development of therapeutics21,22,23. In particular, nanobodies inhibiting the activity of multiple membrane proteins are potential assets for clinical applications5,24,25,26. In the case of membrane transporters, to evaluate whether a nanobody has inhibitory activity, it is necessary to develop an assay that allows the detection of transported substrates and/or co-substrates. Such assays usually involve labeled molecules or the design of substrate-specific detection methods, which may lack a universal application. Furthermore, the identification of inhibitory nanobodies generally requires the screening of large numbers of binders. Thus, a method that can be used in a high-throughput mode and that does not rely on labeled substrates is essential for this selection.
SSM-based electrophysiology is an extremely sensitive, highly time-resolved technique that allows the detection of movement of charges across membranes (e.g., ion binding/transport)27,28. This technique has been applied to characterize electrogenic transporters, which are difficult to study using other electrophysiology techniques due to the relative low turnover of these proteins29,30,31,32,33,34,35. SSM electrophysiology does not require the use of labeled substrates, it is suitable for high-throughput screening, and either proteoliposomes or membrane vesicles containing the transporter of interest can be used. Here, we demonstrate that SSM-based electrophysiology can be used to classify transporter-targeted nanobodies with inhibitory and non-inhibitory properties. As a proof-of-principle, we describe the reconstitution of a bacterial choline transporter into liposomes, followed by detailed steps for immobilization of proteoliposomes on the SSM sensors. We next describe how to perform SSM-based electrophysiology measurements of choline transport and how to determine the half-maximal effective concentration (EC50). We then show how to use SSM-based electrophysiology to screen multiple nanobodies and to identify inhibitors of choline transport. Finally, we describe how to determine the half maximal inhibitory concentrations (IC50) of selected inhibitory nanobodies.

<table><tbody><tr><td>1-octadecanethiol solution</td><td>Sigma Aldrich</td><td>O1858-25ML</td><td></td></tr><tr><td>1,2-diphytanoyl-sn-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850356C-25mg</td><td></td></tr><tr><td>Bio-Beads SM-2 Adsorbent (Polystyrene adsorbent beads)</td><td>BioRad</td><td>#152-3920</td><td></td></tr><tr><td>PD 10 Desalting Columns</td><td>GE Healthcare</td><td>GE17-0851-01</td><td></td></tr><tr><td>Filter 200 nm membrane</td><td>Whatman Nucleopore</td><td>WHA800282</td><td></td></tr><tr><td>2-Propanol</td><td>Merck</td><td>33539-1L-R</td><td></td></tr><tr><td>n-Decane</td><td>Sigma Aldrich</td><td>8034051000</td><td></td></tr><tr><td>n-dodecyl-&amp;szlig;-D-maltoside (DDM)</td><td>Avanti Polar Lipids</td><td>850520P-25g</td><td></td></tr><tr><td>Sodium Chloride</td><td>AppliChem</td><td>131659.1211</td><td></td></tr><tr><td>(SSM setup) SURFE2R N1</td><td>Nanion</td><td>-----</td><td></td></tr><tr><td>SURFE2R N1 Single Sensor Chips</td><td>Nanion</td><td># 161001</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma Aldrich</td><td>T1503</td><td></td></tr><tr><td>E. coli Polar Lipid Extract</td><td>Avanti Polar Lipids</td><td>100600C</td><td></td></tr><tr><td>Egg PC L-&amp;alpha;-phosphatidylcholine </td><td>Avanti Polar Lipids</td><td>840051C</td><td></td></tr></tbody></table>

selection of transporter-targeted inhibitory nanobodies by solid-supported-membrane (ssm)-based electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

SSM-based electrophysiology has been extensively used for the characterization of electrogenic transporters. In the protocol presented here, we show how to use SSM-based electrophysiology to classify nanobodies targeting a secondary transporter (here a bacterial choline symporter) based on their inhibitory and non-inhibitory properties. One of the most useful features of this technique is that it allows for the high-throughput screening of multiple buffer conditions. This particular characteristic is beneficial for the analysis of nanobody libraries, which after the selection of binders can be constituted from a few to dozens of nanobodies. In a standard experiment, a stable lipid monolayer is assembled on a sensor chip. After applying the proteoliposomes preparation containing the choline transporter, a check for good conductivity and capacitance is performed as this is essential for the success of the experiment. In case that the integrity of the membrane is compromised during an experiment, which is easily observed due to the high noise background currents, changing to a new chip is recommended as recovering low noise conditions is rather difficult. In general, we have observed very good reproducibility among measurements of transport and inhibition by nanobodies when using different chips.
To decide about the substrate concentration to be used during a screening of nanobodies, electrogenic transport was first measured under different substrate concentrations to determine EC50 (Figure 1B,C). A substrate concentration that corresponds to saturating conditions was selected (Figure 1C). This substrate concentration was then kept constant in all activating buffers. For this particular example, we selected 5 mM choline.
For the screening of nanobodies, the nanobody must be added to both non-activating and activating buffers. When nanobodies were added to only the activating buffer, it was not possible to observe inhibition of the electrogenic transport. We speculate that this is due to an incomplete occupation of all nanobody binding sites in the transporter population on the chip, thereby revealing the importance of pre-incubation with nanobodies in non-activating conditions. To ensure that all sites are likely to be occupied, a time delay step was included during the application of the first non-activating buffer step to allow the saturation of nanobody binding sites on the transporter population. Incubation times ranging from 2-60 min have been tested with reproducible results. Keep in mind that optimal times of incubation depend on the nature of the nanobody binder and its concentration during the experiment (as well as the concentration of transporter in proteoliposomes on the chip). Therefore, it is recommended to try different incubation times. In any case, as a rule of thumb, the lower the nanobody concentration, the longer the incubation time required. We tested incubation times of 2 min, 20 min, 30 min, and 60 min for different nanobodies but did not detect further transport inhibition.
The effect of inhibitory nanobodies on electrogenic transport is visualized from the decrease of peak currents amplitudes (Figure 2A,C,D). Non-inhibitory nanobodies, on the other hand, do not affect peak currents. After running the washing protocol to allow nanobodies unbinding, a recovery of 80 to 95% of the initial peak current amplitude was observed (Figure 2A,C,D). We have performed a similar experiment but in the presence of liposomes without the transporter protein. When changing from non-activating to activating conditions, no significant artifact currents was introduced by nanobodies present in these buffers (Figure 2B). Running this control experiment is recommended as it is important to know whether changes in peak currents arise from artifacts or not.
After the selection of nanobodies with inhibitory properties, we determined IC50 values for individual nanobodies (Figure 3A,B). For this particular experiment, it is recommended to start with a low concentration of nanobodies and then move towards high concentration during the assay. The calculation of the inhibition for each concentration was then performed by comparing peak currents measured before and after the application of nanobody. To avoid unspecific binding of nanobodies to surfaces, which can be particularly problematic when using low nanobody concentrations, it is advised to follow a similar protocol to that described by Kermani et al.37, where 50 &#181;g/mL of bovine serum albumin was added to the buffers, preventing this deleterious effect. Adding detergents such as Tween or Triton for this purpose should be avoided as these would dissolve lipid membranes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62578/62578fig1v2.jpg" /> 
Figure 1: SSM-based electrophysiology. (A) &#65279;Protocol for transient currents measurement. A non-activating solution is replaced by an activating solution followed by the flow of non-activating solution to restore initial conditions. During the first step, nanobodies bind to the transporter. When switching to the activating solution, the substrate gradient drives the electrogenic transport (orange curve). In the presence of an inhibitory nanobody, the peak current shows a smaller amplitude (blue curve). After finishing the protocol and running solutions without nanobody (wash), unbinding of nanobodies occurs. In the schematic, proteoliposomes with reconstituted protein (blue) are immobilized on the SSM sensor. Triangles and red circles represent nanobodies and substrate, respectively. (B) Electrogenic choline transport in the absence of nanobodies. Peak currents measured during activating conditions are shown for different substrate concentrations. (C) Representative measurement of currents during activating conditions in the absence of transporter protein at different substrate concentrations. (D) Plot of substrate concentration versus peak currents amplitude. The EC50 determined was 95 &#177; 11 &#181;M choline. Error bars indicate standard deviation (n=3 biological replicates, n=3 technical replicates). <a href="https://www.jove.com/files/ftp_upload/62578/62578fig1v2large.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/62578/62578fig2v2.jpg" /> 
Figure 2: Screening and classification of inhibitory and non-inhibitory nanobodies. (A) Electrogenic choline transport in the presence of a nanobody. Peak currents measured during activating conditions are shown in the absence of nanobody (blue), in the presence of an inhibitory nanobody (red), and after nanobody unbinding (green). (B) Measurement of currents during activating conditions in the absence of transporter protein. Traces show recordings in the absence of nanobody (blue), in the presence of an inhibitory nanobody (green), and in the presence of a non-inhibitory nanobody (red). (C,D). Histograms showing peak currents measured during activating conditions in the presence of nanobodies and after nanobody unbinding (recovery). Panel C shows the results of measurements using individual chips per nanobody. Panel D shows the results from a serial measurement using one chip. Nanobodies are indicated as Nb. Error bars indicate the standard deviation (n=3 biological replicates, n=2 technical replicates). <a href="https://www.jove.com/files/ftp_upload/62578/62578fig2v2large.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/62578/62578fig03.jpg" /> 
Figure 3: Determination of IC50 of an inhibitory nanobody. (A) Electrogenic choline transport and inhibition by a nanobody. Peak currents measured during activating conditions are shown for different nanobody concentrations. (B) Plot of peak currents amplitude vs nanobody concentration from a serial measurement with an inhibitory nanobody. The IC50 determined was 18 &#177; 2 nM. Error bars indicate the standard deviation (n=3 biological replicates, n=3 technical replicates). <a href="https://www.jove.com/files/ftp_upload/62578/62578fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology at JoVE.com

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

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Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.5K Views.  Biozentrum, University of Basel. Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters. 

Video: Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Bioengineering

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Neuroscience

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

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

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

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

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

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

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Chapters in this video

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Introduction to Solid Supported Membrane Based Electrophysiology

Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

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

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge