monitoring the arrival of gfp-tagged surface proteins to the trans-golgi network using functionalized nanobodies

Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies

In a cell culture hood, seed between 400,000 and 500,000 HeLa cells that stably express a GFP-tagged reporter protein into either 35mm dishes or 6-well clusters, with complete medium containing antibiotics. Incubate the cells at 37 degrees Celsius with 7.5% carbon dioxide overnight. The next day, remove the complete medium, and wash the cells twice with 1X PBS at room temperature. Starve the cells with sulfate-free medium for 1 hour at 37 degrees Celsius with 7.5% carbon dioxide.
Meanwhile, in a ventilated hood or bench designated for radioactivity work, prepare sulfate labeling medium containing 0.5 millicuries per milliliter sodium sulfate S35 in sulfate-free medium. Next, mix VHH double-tier-sulf in 1X PBS with sulfate labeling medium to a final concentration of 2 micrograms per milliliter. When the incubation is complete, replace the sulfate-free media with 0.7 milliliters of the prepared medium mixture, containing sodium sulfate S35 and VHH double-tier-sulf. Incubate the cells for 1 hour at 37 degrees Celsius in a 7.5% carbon dioxide incubator, designed for work with radioactivity.
After this, remove the labeling medium, and wash the cells two to three times with ice-cold 1X PBS on either a cooling plate or on ice. Add 1 milliliter of lysis buffer, supplemented with two millimolar PMSF and 1X protease inhibitor cocktail. Incubate the cells for 10 to 15 minutes on a rocking platform at 4 degrees Celsius. Then, scrape and transfer the lysate to a new 1.5-milliliter tube, vortex the lysate, and place it on an end-over-end rotator at 4 degrees Celsius for 10 to 15 minutes. Centrifuge at 10,000 g and at 4 degrees Celsius for 10 minutes to prepare a post-nuclear lysate. Transfer the post-nuclear lysate into a 1.5-milliliter tube containing prepared nickel beads, and incubate on an end-over-end shaker at 4 degrees Celsius for 1 hour to isolate the nanobodies.
After this, wash the beads three times with either 1X PBS or lysis buffer by gently pelleting at 1000 g for 1 minute. Carefully remove all of the washing buffer from the beads, and add 50 microliters of 2X sample buffer. Boil at 95 degrees Celsius for 5 minutes. Then, load the boiled beads on a midi 12.5% SDS-PAGE gel, and run according to the standard PAGE protocol until the reference dye has reached the end of the separating gel. Fix the separating gel in approximately 30 milliliters of fixation buffer for 1 hour at room temperature. Wash the gel three times with approximately 50 milliliters of deionized water, and then dry the gel, as outlined in the text protocol. When the drying is complete, use a phosphor screen imager to image autoradiograph the gel.

monitoring-arrival-gfp-tagged-surface-proteins-to-trans-golgi-network

Universidad San Sebastian

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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

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1. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis)
<ol>
	<li>In a cell culture hood, seed ~400,000 to 500,000 HeLa cells stably expressing a GFP-tagged reporter protein in 35 mm dishes or on 6-well clusters with complete medium containing antibiotics (Dulbecco&#39;s Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 &#181;g/mL puromycin). 
	NOTE: As mentioned above, we here use HeLa cells stably expressing EGFP-Calnexin, EGFP-CDMPR, and TfR-EGFP for illustration purposes. Apart from stable cell lines, also transiently transfected HeLa can be used.</li>
	<li>Incubate the cells O/N at 37 &#176;C in a 7.5% CO2 incubator to proliferate. 
	NOTE: Cells should have a confluency of ~80% the next day.</li>
	<li>Remove the complete medium, wash 2 times with 1x PBS at RT, and starve the cells with sulfate-free medium (SFM) for 1 h at 37 &#176;C in a 7.5% CO2 incubator. 
	NOTE: SFM is prepared by adding 10 mL of MEM amino acids (50x) solution, 5 mL of L-glutamine (200 mM), 5 mL of vitamin solution (100x), and 900 &#181;L of CaCl2&#183;2H2O to 500 mL of Eagle&#39;s balanced salts. Pass through a 0.22 &#181;m filter and aliquot.</li>
	<li>In a ventilated hood or bench designated for radioactivity work, prepare a sulfate labeling medium containing 0.5 mCi/mL [35S] sulfate as a sodium salt in SFM. 
	CAUTION: Carefully handle all solutions containing radioactivity. Only work in designated hoods or benches. All material (tips, tubes, plates, etc.) or wash buffers in contact with radioactivity have to be collected and disposed of separately. Do this for all steps below (steps 1.5-1.18) as well.</li>
	<li>Add VHH-2xTS in 1x PBS into sulfate labeling medium to a final concentration of 2 &#181;g/mL. 
	NOTE: As a control, also include VHH-std or another nanobody devoid of TS sites to demonstrate specific labeling.</li>
	<li>Replace SFM with 0.7 mL of sulfate labeling medium containing 0.5 mCi/mL [35S]sulfate and 2 &#181;g/mL VHH-2xTS and incubate the cells for 1 h at 37 &#176;C in a 7.5% CO2 incubator designated for work with radioactivity. 
	NOTE: Depending on the planned experiment, the time of nanobody sulfation needs to be adjusted. In addition, to determine TGN arrival kinetics, labeling is performed for different times (e.g., for 10 min, 20 min, 30 min, etc.).</li>
	<li>Remove the labeling medium and wash the cells 2-3 times with ice-cold 1x PBS on a cooling plate or ice.</li>
	<li>Add 1 mL of lysis buffer (PBS containing 1% Triton X-100 and 0.5 % deoxycholate) supplemented with 2 mM PMSF and 1x protease inhibitor cocktail and incubate the cells for 10-15 min on a rocking platform at 4 &#176;C.</li>
	<li>Scrape and transfer the lysate into a 1.5 mL tube, vortex the lysate, and place for 10-15 min on an end-over-end rotator at 4 &#176;C.</li>
	<li>Prepare a postnuclear lysate by centrifugation at 10,000 x g for 10 min at 4 &#176;C.</li>
	<li>Using a new 1.5 mL tube, prepare 20-30 &#181;L of a slurry of Nickel beads in a 1.5 mL tube and wash once with 1x PBS by gently pelleting them at 1,000 x g for 1 min. 
	NOTE: Instead of Nickel beads, streptavidin beads (if the nanobody is biotinylated) or protein A beads with an IgG against an epitope of the nanobody (T7- or HA-tag) can be used.</li>
	<li>Transfer the postnuclear lysate into a 1.5 mL tube containing Nickel beads and incubate for 1 h at 4 &#176;C on an end-over-end rotator to isolate the nanobodies. Remove an aliquot (50-100 &#181;L) of the postnuclear lysate and boil in SDS sample buffer for subsequent immunoblot analysis of total cell-associated nanobody, GFP reporter, and loading control.</li>
	<li>Wash the beads 3 times with 1x PBS or lysis buffer by gently pelleting at 1,000 x g for 1 min. 
	NOTE: For more stringent washing of beads, 20 mM imidazole can be included in PBS or lysis buffer.</li>
	<li>Carefully remove all washing buffer from the beads, add 50 &#181;L of 2x sample buffer, and boil for 5 min at 95 &#176;C.</li>
	<li>Load both boiled beads on a midi 12.5% SDS-PAGE gel and run according to the standard PAGE protocol until the reference dye (e.g., bromophenol blue) has reached the end of the separating gel. 
	NOTE: Immunoblot analysis of total cell-associated nanobody, GFP reporter, and loading control can also be performed using a mini 12.5% SDS-PAGE or precast gradient gel.</li>
	<li>Fix the separating gel in ~30 mL of fixation buffer (10% acetic acid and 45% methanol in ddH2O) for 1 h at RT, wash the gel three times with ~50 mL of deionized water, and prepare for gel drying.</li>
	<li>Drying SDS-PAGE Gels.&#8203;
	<ol>
		<li>Carefully place the fixed gel on stretched cling film and cover it with precut filter paper.</li>
		<li>Place the gel with the filter paper down on top of the drying platform, place the vacuum cover, and switch on the vacuum pump for gel drying. Dry the gel for 3-5 h.</li>
		<li>Remove the cling film and place the dried gel into an autoradiography cassette equipped with a Phosphor Screen.</li>
	</ol>
	</li>
	<li>Image autoradiograph using a Phosphor Screen Imager. Follow the instructions of the manufacturer. 
	NOTE: Depending on the strength of the signal, dried gels need to be exposed longer with Phosphor screens.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Sigma-Aldrich</td>
			<td>1.18145E+11</td>
			<td>Product is distributed by SigmaAldrich, but manufactured by Roche</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biowest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfur-35 as sodium sulfate</td>
			<td>Hartmann Analytics</td>
			<td>ARS0105</td>
			<td>Product contains 5 mCi</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>TPP</td>
			<td>92406</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Applichem</td>
			<td>A3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Protean TGX gels, 4-20%, 15- well</td>
			<td>Bio-Rad</td>
			<td>456-1096</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Buser, D. P., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59111">Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells.</a> J. Vis. Exp. (2019).
This video demonstrates a method to study the uptake and retrograde transport of functionalized nanobodies to understand their intracellular trafficking pathways. By introducing radiolabelled sulfate and utilizing isolation and detection techniques, this method enables the study of endocytic uptake and trans-Golgi network arrival of cargo proteins.

1. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis)

	In a cell culture hood, seed ~400,000 to 500,000 HeLa cells stably ...

Start with sulfate-deprived cancer cells expressing a GFP-tagged surface protein.
Add radiolabeled sulfate and modified anti-GFP nanobodies &#8212; a single-chain antibody carrying a tyrosine-sulfation motif, and incubate.
Sulfate-deprived cells internalize radiolabeled sulfates, incorporating them into cellular components.
The anti-GFP nanobody binds to the GFP-tagged surface protein and gets endocytosed into a vesicle. This vesicle transports the nanobody-surface protein complex along specific intracellular pathways, including the trans-Golgi network, or TGN.
Within the TGN, the sulfotransferase in the presence of a substrate transfers a radioactive sulfate to specific tyrosine residues on the nanobody, thereby radiolabeling it.
Wash the cells. Next, lyse the cells.
Centrifuge the lysate to remove large cellular debris. Isolate the nanobodies using a nickel bead-based purification method.
Load the isolated fraction on an SDS-PAGE gel to separate the radiolabeled nanobody into distinct bands.
Subject the gel to autoradiography.
The resulting autoradiograph reveals darkened regions, indicating the presence of the radiolabeled nanobody.

18 Views. Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies

Video: Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies - Experiment

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.

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.

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

JoVE Journal

Biochemistry

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Neuroscience

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies

Transcript

Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies