Name: determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation
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Description: Watch this Scientific Journal Video about Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation at JoVE.com

This project was supported by the Canadian Institute of Health Research PG#20R47867.

1. Determining Relative Cell-surface Protein Expression in Astrocytes by Biotinylation
NOTE: Here, we illustrate the application of this biotinylation technique to the study of the effects of the extracellular matrix molecule laminin on the cell-surface localization of the water-permeable channel aquaporin-4 (AQP4). Specialized materials required for this assay include sulfo-NHS-LC-biotin and streptavidin-agarose resin (see Table of Materials).
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Modifications: 
As these methods were designed for use with adherent cells, we have specified the use of PBS containing 100 mg/L MgCl2&#8729;6H2O and
100 mg/L CaCl2 (CM-PBS) for the washing steps and as the base of certain buffers so as to ensure that the cells remain attached to the culture surface and that cell-cell junctions are not disrupted. However, the protocols may also be applied to nonadherent cell types if the cells are pelleted...

Proteins at the cell surface play a variety of roles central to maintaining cell function. In numerous instances, their activity is dependent on, or modulated by endocytic processes that either temporarily sequester them in intracellular sites, or that direct them towards degradative pathways1,2,3,4,5. Here, we highlight 2 biotinylation-based approaches designed to allow the user to specifically tag and isolate proteins expressed at the plasma membrane, and those newly internalized. Via these methods, the cell-surface expression and endocytic rate of any protein of interest may be quantified, thus allowing a clearer assessment of its regulation to be achieved.
Determining relative cell-surface protein expression by biotinylation
Biotin, or vitamin B7, formerly known as vitamin H6, is a small water soluble molecule that can be used to chemically modify reactive amine, sulfhydryl, and carboxyl groups of biological molecules. The current crop of cell-surface biotinylation reagents consist primarily of membrane-impermeant sulfonated N-hydroxysuccinimide (sulfo-NHS) esters of biotin or its derivatives, designed to react with the amines present on the side-chains of lysine residues of proteins expressed at the cell surface when these become deprotonated under basic conditions, resulting in the latter forming an amide bond with the biotin moiety7. Thusly modified, cell-surface proteins may then be isolated via the use of avidin, a 66 - 69 kDa tetrameric protein possessing great affinity for biotin, binding to the latter with a dissociation constant of approximately 10-15, marking it as one of the strongest noncovalent interactions known8,9.
A number of alternative methods of quantifying protein expression at the cell surface have been used in previous studies. The labeling of unpermeabilized cells using fluorescently tagged antibodies specific for the protein of interest, followed by visualization via fluorescence microscopy, for instance, is a commonly employed approach, but is heavily reliant on the availability of antibodies that can bind to extracellular epitopes. More recently, methods involving the use of chimeric proteins bearing pH-sensitive fluorophores that react to being exposed to acidic media have also been successfully employed10. However, such assays usually involve the exogenous expression of these constructs in cell lines in which the protein of interest is not natively found. These approaches are nonetheless able to provide valuable information regarding the subcellular localization and exocytic itinerary of the target protein, and should therefore be used in conjunction with the biotinylation-based approaches described here if the tools are available.
In a typical biotinylation assay, the cells are first washed thoroughly in 4 &#176;C PBS. This removes any traces of serum proteins introduced by the culture medium, thus ensuring that these will not consume excess amounts of biotin in the next step. More importantly, the reduction in temperature causes endocytosis to decelerate significantly. The biotinylation reagent is then added. Next, the cells are washed again, and then incubated with a quenching buffer containing either glycine or NH4Cl, the purpose of which is to inactivate all remaining traces of unreacted biotin. The cells are then lysed, following which agarose-immobilized streptavidin is added to precipitate the biotinylated proteins. Analysis is commonly performed via western blotting, allowing the relative cell-surface expression of various proteins to be quantified.
Due to the basis of this assay, it is suitable for use only with proteins possessing portions exposed to the extracellular environment. Multipass transmembrane proteins, which likely possess a number of reactive lysines within their loop regions, are the most amenable to this method, while single-pass proteins tend to be less susceptible to being biotinylated. Even in these cases, there remains a possibility that conformational changes or intermolecular interactions may occlude certain reactive sites, resulting in a lower-than-expected biotinylation yield.
Determining internalization rate of cell-surface proteins by biotinylation
The principles of this assay are largely similar to those of cell-surface biotinylation, with a number of exceptions, the most important of which is the use of reversible biotinylation reagents. The biotin groups (of these) possess disulfide bonds, within their structures, that are susceptible to reducing agents; this is exploited to ensure that only cell-surface proteins taken into intracellular sites during the assay period will be left biotinylated. An assay generally takes place in the following manner. The cells are first washed and biotinylated with cold reagents, then cell culture medium at 37 &#176;C is re-introduced, and the cells are returned to the incubator; this causes the labeled cell-surface proteins to undergo endocytosis. The reducing agent glutathione - which cannot penetrate the membrane - is then added to break the disulfide bonds of the biotin moieties attached to proteins remaining on the cell surface. Finally, the broken disulfide bonds are reacted with iodoacetamide, consuming the labile thiol groups and preventing the bonds from reforming. As before, the cells are then lysed, and the labeled proteins are precipitated using streptavidin-agarose.
The limitations discussed in the previous section also apply here due to the similarities shared between the methods. In addition, it is worth bearing in mind that the temperature shifts involved in this assay preclude the exact determination of how much protein is endocytosed for each increment of time, particularly in the case of rapidly internalized or rapidly recycling proteins. The assay therefore only provides a semiquantitative estimation of endocytic rates. Total internal reflection fluorescence microscopy can be used to track the uptake of each loaded vesicle and provide a more precise measurement of the kinetics of endocytosis. It can therefore provide a very useful complement to this assay, assuming that a fluorescently tagged chimeric construct of the protein of interest is available11.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Ammonium chloride (NH4Cl)</td>
			<td style="width:219px;">Fisher Scientific</td>
			<td style="width:129px;">A661-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>#1610404</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">cOmplete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Disodium ethylenediaminetetraacetate dihydrate (EDTA)</td>
			<td>Bio-Rad</td>
			<td style="width:129px;">#1610729</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Dithiothreitol (DTT)</td>
			<td>Bio-Rad</td>
			<td>#1610611</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21335</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-SS-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21331</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Fetal bovine serum</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Iodoacetamide</td>
			<td>Bio-Rad</td>
			<td>#163-2109</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Thaw on ice.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">L-glutamine</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-actin antibody (AC-15)</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-dystroglycan antibody (43DAG1/8D5)</td>
			<td>Leica Biosystems</td>
			<td>B-DG-CE</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Penicillin/streptomycin</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>715-035-150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-035-045</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Phosphate buffer saline</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Reduced glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G6529</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>862010&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Streptavidin agarose resin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#20347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Rabbit polyclonal anti-AQP4 antibody</td>
			<td>Alomone</td>
			<td>AQP-004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Tris base (Trizma base)</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using cell-surface biotinylation to assess the plasma membrane expression of AQP4 in astrocytes 
Laminin-treated astrocyte cultures and untreated control cells were subject to cell-surface biotinylation using the methods described. Biotinylated proteins were precipitated with agarose-conjugated streptavidin, and then separated via SDS-PAGE. Cell-surface fractions were probed for AQP4&#160;and &#946;-dystroglycan (&#946;-DG) as a cell-surface loading control, while th...

Watch this Scientific Journal Video about Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation at JoVE.com

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

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 ...

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