Name: Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation (Video) | JoVE Journal
Uploaded: 2017-07-03T15:00:00.000+00:00
Duration: 10 min 42 s
Description: Learn cell-surface and pulse-chase biotinylation methods to study Aquaporin-4 expression and endocytic rates in primary astrocyte cultures. These techniques reveal protein dynamics at the plasma membrane.

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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).
<ol>
	<li>Using the method outlined in Noel et al.12, prepare cultures of cortical astrocytes approximately 2 weeks in advance of the assay, and grow them in 75 cm2 vented culture flasks. When astrocytes are 80 - 90% confluent, detach them from the culture surface using 0.05% trypsin, and then passage them 1:3.</li>
	<li>At 48 h prior to the assay, when cells are again 80 - 90% confluent, passage astrocytes 1:3 (accounting for the change in the culture format) onto 60 mm cell culture dishes so that they are &#62;70% confluent on the day the experiment is to take place. Ensure that cells are evenly distributed between dishes.</li>
	<li>At 16 h prior to assay, pipette laminin into culture medium to a final concentration of 24 nM, and incubate at 37 &#176;C.</li>
	<li>Immediately prior to assay, prepare the following, and then place on ice or refrigerate: CM-PBS (100 mg/L MgCl2&#8729;6H2O and 100 mg/L CaCl2 in 1X PBS, pH 7.4), biotin buffer (0.5 mg/mL sulfo-NHS-LC-biotin in CM-PBS), quenching buffer (50 mM NH4Cl in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM DTT and 0.01% bromophenol blue), and wash buffer (10 mM Tris (pH 7.4), 1.5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1X protease inhibitor cocktail).</li>
	<li>Remove dishes holding the astrocyte cultures from the incubator, and discard the medium.</li>
	<li>Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.</li>
	<li>Pipette 2 mL biotin buffer into each well, and gently tilt dishes back and forth a few times to ensure complete coverage. Leave on ice for 30 min.</li>
	<li>Remove the biotin buffer using an aspirator, and replace with 4 mL quenching buffer. Leave on ice for 10 min.</li>
	<li>Aspirate the quenching buffer, and replace with an equivalent volume of the same. Again, leave on ice for 10 min.</li>
	<li>Discard the quenching buffer, and wash cells thrice with 4 mL chilled CM-PBS.</li>
	<li>Scrape cells into 1 mL chilled CM-PBS using a cell lifter, and transfer the suspension to microcentrifuge tube.</li>
	<li>Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and re-suspend cells in 500 &#181;L of lysis buffer.</li>
	<li>Leave samples on ice for 30 min, vortexing every 5 min, or place them on an end-over-end rotator at 4 &#176;C.</li>
	<li>Centrifuge the lysate at 14,000 x g for 10 min at 4 &#176;C to pellet any detergent-insoluble materials. Transfer the supernatant into a new microcentrifuge tube.
	<ol>
		<li>Save 50 &#181;L of this lysate and add loading buffer to it. Then denature it by heating at 95 &#176;C in a dry bath; this is the &#34;input&#34; fraction, containing both biotinylated cell-surface proteins, as well as non-biotinylated cytosolic proteins.</li>
	</ol>
	</li>
	<li>Widen the opening of a pipette tip by cutting off approximately 0.5 cm of material from its end using a pair of sharp scissors. Using this pipette tip, transfer 75 &#181;L of streptavidin-agarose beads (normally stored at 4 &#176;C) to the lysate, and incubate at 4 &#176;C for 3 h on shaker/rocker.
	<ol>
		<li>As streptavidin-agarose is frequently sold as a slurry containing 50% beads by volume, suspended in an antimicrobial solution, triturate the slurry to ensure that the beads are evenly suspended, and then pipette 150 &#181;L of the suspension into each sample.</li>
	</ol>
	</li>
	<li>Pellet streptavidin-agarose beads by centrifugation at 1500 x g for 30 s at 4 &#176;C.
	<ol>
		<li>Save 50 &#181;L of the supernatant (add loading buffer and denature it at 95 &#176;C in a water bath or heating block); this represents the &#34;intracellular&#34; fraction, and is comprised primarily of non-biotinylated cytosolic proteins.</li>
	</ol>
	</li>
	<li>Resuspend the pelleted beads in 1 mL wash buffer, and rock this for 3 min at 4 &#176;C. Pellet beads (as in step 1.16), and discard the supernatant. Repeat this process 4x to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.</li>
	<li>Pellet the beads by centrifugation (1500 x g for 30 s at 4 &#176;C), and discard the overlying wash buffer. Add 50 &#181;L of 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing this at 95 &#176;C; this fraction should contain biotinylated cell-surface proteins only (&#34;cell-surface&#34; fraction).
	<ol>
		<li>Separate input, cell-surface, and intracellular fractions by SDS-PAGE 13, and analyze by western blotting 14. 
		NOTE: While we used a 4 - 20% precast gradient gel in our experiments, a 12 - 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.</li>
	</ol>
	</li>
</ol>
2. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation
NOTE: In the following, we describe a&#160;typical pulse-chase biotinylation experiment&#160;used in this instance to track the endocytosis of AQP4 in astrocytes. This method is based on that used by Madrid et al.15. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see the Table of Materials).
<ol>
	<li>Prepare cultures of mouse cortical astrocytes in 60 mm dishes using the methods outlined in the previous section. Ensure that cells are approximately 70% confluent on the day of the assay, and that each dish contains an equivalent number of cells.</li>
	<li>Immediately prior to assay, prepare the following, and place on ice or refrigerate: CM-PBS (100 mg/L MgCl2&#8729;6H2O and 100 mg/L CaCl2 in 1X PBS, pH7.4), biotin buffer (0.5 mg/mL sulfo-NHS-SS-biotin in CM-PBS), reducing buffer (50 mM reduced glutathione, 75 mM NaCl and 75 mM NaOH), quenching buffer (50 mM iodoacetamide, 1% BSA, in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM DTT and 0.01% bromophenol blue), and wash buffer (10 mM Tris, pH 7.4, 1.5 mM EDTA, 150 mM NaCl, 1% triton X-100, 1X protease inhibitor cocktail).</li>
	<li>Prepare fresh cell culture medium (DMEM supplemented with 10% Fetal Bbovine Serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine), and place it in a 37 &#176;C water bath.</li>
	<li>Remove astrocyte cultures from the incubator, and aspirate the medium using an aspirator.</li>
	<li>Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.</li>
	<li>Pipette 3 mL biotin buffer into each dish, tilt dishes back and forth a few times to ensure that buffer is well-distributed, and leave this on ice for 30 min.</li>
	<li>Aspirate biotin buffer, and replace it with 5 mL warm medium. Incubate a culture dish at 37 &#176;C for 15 min, and a second dish at the same temperature for 30 min. Leave another dish at 4 &#176;C as the 0 min sample.</li>
	<li>At the end of the incubation period, discard medium, and wash cells thrice with 4 mL chilled CM-PBS. Pipette 6 mL reducing buffer over cells, and leave on ice for 15 min.</li>
	<li>Remove reducing buffer, and then replace with 6 mL fresh reducing buffer.&#160;Place on ice for an additional 15 min.</li>
	<li>Remove reducing solution and replace with 6 mL quenching buffer. Leave on ice for 15 min.</li>
	<li>Repeat quenching step once more.</li>
	<li>Discard quenching buffer, and wash cells thrice with 4 mL chilled PBS.</li>
	<li>Scrape cells into 1 mL chilled PBS using a cell lifter, and transfer the suspension into a microcentrifuge tube.</li>
	<li>Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and resuspend cells in 500 &#181;L lysis buffer.</li>
	<li>Leave this on ice for 30 min and vortex every 5 min. Alternatively, place the samples on an end-over-end rotator at 4 &#176;C for this duration.</li>
	<li>Centrifuge the lysate at 14,000 x g for 10 min at 4 &#176;C to pellet the detergent-insoluble materials, then transfer supernatant to a new microcentrifuge tube. Save 50 &#181;L of this lysate, add loading buffer to it and denature at 95 &#176;C in a dry bath; this is the &#34;input&#34; fraction, containing both biotinylated endocytosed proteins, as well as nonbiotinylated proteins.</li>
	<li>Using a cut pipette tip, add 150 &#181;L of the streptavidin-agarose slurry to the lysate, and incubate at 4 &#176;C for 3 h on shaker/rocker. See 1.15 for additional details on this step.</li>
	<li>Pellet streptavidin-agarose beads by centrifugation at 1,500 x g for 30 s at 4 &#176;C.</li>
	<li>Resuspend beads in 1 mL wash buffer, and rock for 3 min at 4 &#176;C. Pellet beads (as per step 2.18), and discard the supernatant. Repeat this process 4x to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.</li>
	<li>Pellet beads by centrifugation at 1,500 x g for 30 s, at 4 &#176;C, and discard overlying wash buffer. Add 50 &#181;L 1X loading buffer (diluted&#160;using lysis buffer). Release biotin and streptavidin from beads by denaturing at 95 &#176;C; this fraction should contain internalized cell-surface proteins only (&#34;endocytosed&#34; fraction).</li>
	<li>Separate input, cell-surface, and unbound fractions by SDS-PAGE13, and analyze by western blotting14. 
	NOTE: While we used a 4 - 20% precast gradient gel in our experiments, a 12 - 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.</li>
</ol>

<ol><li>Baskin, G., Schenker, S., Frosto, T., Henderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transforming+growth+factor+beta+1+inhibits+epidermal+growth+factor+receptor+endocytosis+and+down-regulation+in+cultured+fetal+rat+hepatocytes%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes.</a> J Biol Chem. 266 (20), 13238-13242 (1991).</li>
<li>Hazum, E., Cuatrecasas, P., Marian, J., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Receptor-mediated+internalization+of+fluorescent+gonadotropin-releasing+hormone+by+pituitary+gonadotropes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes.</a> Proc Natl Acad Sci U S A. 77 (11), 6692-6695 (1980).</li>
<li>Hemmaplardh, D., Morgan, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transferrin+uptake+and+release+by+reticulocytes+treated+with+proteolytic+enzymes+and+neuraminidase%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.</a> Biochim Biophys Acta. 426 (3), 385-398 (1976).</li>
<li>Katsura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constitutive+and+regulated+membrane+expression+of+aquaporin+1+and+aquaporin+2+water+channels+in+stably+transfected+LLC-PK1+epithelial+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.</a> Proc Natl Acad Sci U S A. 92 (16), 7212-7216 (1995).</li>
<li>Li, Y., Cam, J., Bu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-density+lipoprotein+receptor+family%3A+endocytosis+and+signal+transduction%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">Low-density lipoprotein receptor family: endocytosis and signal transduction.</a> Mol Neurobiol. 23 (1), 53-67 (2001).</li>
<li>Gyorgy, P., Melville, D. B., Burk, D., Du Vigneaud, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+possible+identity+of+vitamin+H+with+biotin+and+coenzyme+R%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The possible identity of vitamin H with biotin and coenzyme R.</a> Science. 91 (2358), 243-245 (1940).</li>
<li>Cole, S. R., Ashman, L. K., Ey, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biotinylation%3A+an+alternative+to+radioiodination+for+the+identification+of+cell+surface+antigens+in+immunoprecipitates%5BTitle%5D)+AND+%22Mol+Immunol%22%5BJournal%5D)">Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates.</a> Mol Immunol. 24 (7), 699-705 (1987).</li>
<li>Green, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Avidin+1.+the+use+of+%5B14-C%5Dbiotin+for+kinetic+studies+and+for+assay%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Avidin 1. the use of [14-C]biotin for kinetic studies and for assay.</a> Biochem J. 89 (3), 585-591 (1963).</li>
<li>Korpela, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Avidin%2C+a+high+affinity+biotin-binding+protein%2C+as+a+tool+and+subject+of+biological+research%5BTitle%5D)+AND+%22Med+Biol%22%5BJournal%5D)">Avidin, a high affinity biotin-binding protein, as a tool and subject of biological research.</a> Med Biol. 62 (1), 5-26 (1984).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+pHluorin-tagged+receptor+insertion+to+the+plasma+membrane+in+primary+cultured+mouse+neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Imaging pHluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons.</a> J Vis Exp. (69), e4450(2012).</li>
<li>Soohoo, A. L., Bowersox, S. L., Puthenveedu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+clathrin-mediated+endocytosis+of+G+protein-coupled+receptors+at+single-event+resolution+via+TIRF+microscopy%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Visualizing clathrin-mediated endocytosis of G protein-coupled receptors at single-event resolution via TIRF microscopy.</a> J Vis Exp. (92), e51805(2014).</li>
<li>Noel, G., Tham, D. K., Moukhles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interdependence+of+laminin-mediated+clustering+of+lipid+rafts+and+the+dystrophin+complex+in+astrocytes%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.</a> J Biol Chem. 284 (29), 19694-19704 (2009).</li>
<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li>
<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets%3A+procedure+and+some+applications%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li>
<li>Madrid, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polarized+trafficking+and+surface+expression+of+the+AQP4+water+channel+are+coordinated+by+serial+and+regulated+interactions+with+different+clathrin-adaptor+complexes%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.</a> EMBO J. 20 (24), 7008-7021 (2001).</li>
<li>Tham, D. K., Joshi, B., Moukhles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aquaporin-4+Cell-Surface+Expression+and+Turnover+Are+Regulated+by+Dystroglycan%2C+Dynamin%2C+and+the+Extracellular+Matrix+in+Astrocytes%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes.</a> PLoS One. 11 (10), 0165439(2016).</li>
</ol>

The authors declare no conflict of interest.

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.

<table><tbody><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647-50</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bio-Rad</td><td>#1610404</td><td></td></tr><tr><td>cOmplete protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>11697498001</td><td></td></tr><tr><td>Disodium Ethylenediaminetetraacetate dihydrate (EDTA)</td><td>Bio-Rad</td><td>#1610729</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-Rad</td><td>#1610611</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco/Thermo Fisher Scientific</td><td>11960-044</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>#21335</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-SS-Biotin</td><td>Thermo Fisher Scientific</td><td>#21331</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898&amp;nbsp;</td><td></td></tr><tr><td>Iodoacetamide</td><td>Bio-Rad</td><td>#163-2109</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich</td><td>L2020</td><td>Thaw on ice.</td></tr><tr><td>L-glutamine</td><td>Gibco/Thermo Fisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-actin antibody (AC-15)</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-dystroglycan antibody (43DAG1/8D5)</td><td>Leica Biosystems</td><td>B-DG-CE</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco/Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>715-035-150</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-035-045</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>Reduced glutathione</td><td>Sigma-Aldrich</td><td>G6529</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma-Aldrich</td><td>862010&amp;nbsp;</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318-100</td><td></td></tr><tr><td>Streptavidin agarose resin</td><td>Thermo Fisher Scientific</td><td>#20347</td><td></td></tr><tr><td>Rabbit polyclonal anti-AQP4 antibody</td><td>Alomone</td><td>AQP-004</td><td></td></tr><tr><td>Tris base (Trizma base)</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP153-1</td><td></td></tr><tr><td>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

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 and β-dystroglycan (β-DG) as a cell-surface loading control, while the input and intra...

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 ...

In this video, two methods will be presented, cell-surface biotinylation, and pulse-chase biotinylation. In the following demonstration, we will be using astrocytes. These are the most abundant glial cell types in the brain, and they happen to express our protein of interest which is Aquaporin-4.The methods that we use in this paper could be applied to many different cell types, and those could include either cells in suspension or adherent cells. These techniques can be used to study the cell surface expression and internalization of virtually any transmembrane protein, as long as it possesses an extracellular domain accessible to biotin. The overall goal of this cell-surface biotinylation assay is to determine the effects of laminin on the expression of the water permeable channel Aquaporin-4 at the plasma membrane of astrocytes.To begin this procedure, remove the astrocyte cultures from the incubator, and discard the medium by aspiration. Wash the cells thrice with four milliliters of chilled CM-PBS, and place the dishes on crushed ice. Remove the CM-PBS by aspiration, and then pipette two milliliters of biotin buffer into each well.Gently tilt the dishes back and forth a few times to ensure complete coverage before placing the cultures on ice for 30 minutes. Afterward remove the biotin buffer, replace it with four milliliters of quenching buffer and keep the cultures on ice for 10 minutes. Next, aspirate and replace with the same amount of the quenching buffer before leaving the cultures on ice for 10 minutes.Then, discard the quenching buffer and wash the cells thrice with four milliliters of chilled CM-PBS. Following this, scrape the cells into one milliliter of chilled CM-PBS and transfer the suspension to a microcentrifuge tube. Next, pellet the cells by centrifugation at 100 g for three minutes in a refrigerated microcentrifuge set to four degrees Celsius.After that, discard the supernatant and resuspend the cells in 500 microliters of lysis buffer. Then, leave the samples on ice for 30 minutes, vortexing every five minutes. Following that, centrifuge the lysate at 14, 000 g for 10 minutes at four degrees Celsius to pellet any detergent and soluble materials.Then, transfer the supernatant into a new microcentrifuge tube. Save 50 microliters of the lysate, and add 25 microliters of three X loading buffer to it. Subsequently, denature it by heating at 95 degrees Celsius in a dry bath.This is the input fraction, containing both biotinylated cell-surface proteins as well as nonbiotinylated cytosolic proteins. Using a cut pipette tip, transfer 75 microliters of streptavidin agarose beads to the lysate, and incubate it at four degrees Celsius for three hours on a shaker. After that, pellet the streptavidin agarose beads by centrifugation at 1, 500 g for 30 seconds at four degrees Celsius.Save 50 microliters of the supernatant and add 25 microliters of three X loading buffer. Then, denature it at 95 degrees Celsius in a water bath or a heating block. This represents the intracellular fraction, and is comprised primarily of nonbiotinylated cytosolic proteins.Afterward, resuspend the pelleted beads in one milliliter of wash buffer and rock it for three minutes at four degrees Celsius. Pellet the beads and discard the supernatant. Repeat this process four more times to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.Add 50 microliters of loading buffer diluted to one X using lysis buffer. Release biotin and streptavidin from the beads by denaturing them at 95 degrees Celsius. This fraction should contain biotinylated cell-surface proteins only.Subsequently, separate the input, cell surface, and intracellular fractions by SDS-PAGE and analyze by western blotting. The overall goal of the pulse-chase biotinylation experiment is to determine the approximate internalization rate of Aquaporin-4 in astrocytes. First, remove the astrocyte cultures from the incubator and aspirate the medium.Then, wash the cells thrice with four milliliters of chilled CM-PBS and place the dishes on crushed ice. Aspirate the CM-PBS, and then pipette three milliliters of biotin buffer into each dish. Tilt the dishes back and forth a few times to ensure that buffer is well-distributed and leave them on ice for 30 minutes.Then, aspirate the biotin buffer, and replace it with five milliliters of warm medium. Incubate one culture dish at 37 degrees Celsius for 15 minutes and a second dish at the same temperature for 30 minutes. Leave another dish at four degrees Celsius as the zero-minute sample.At the end of the incubation period, discard the medium and wash the cells thrice with four milliliters of chilled CM-PBS. Afterwards, aspirate the CM-PBS, and then pipette six milliliters of reducing buffer over the cells, and leave the sample on ice for 15 minutes. Then, replace the medium with six milliliters of fresh reducing buffer, and place the sample on ice for an additional 15 minutes.This step is critical as the glutathione contained within the reducing buffer will cleave the biotin moasis remaining at the cell surface. This ensures that only internalized proteins will be biotin labeled. Following that, remove the reducing solution, and replace it with six milliliters of quenching buffer.Leave the sample on ice for another 15 minutes, and repeat the quenching step once more. Then, discard the quenching buffer, and wash the cells thrice, with four milliliters of chilled PBS. Aspirate the CM-PBS, and then scrape the cells into one milliliter of chilled PBS, using a cell lifter, and transfer the suspension into a microcentrifuge tube.Pellet the cells by centrifugation at 100 g for three minutes. After three minutes, discard the supernatant, and resuspend the cells in 500 microliters of lysis buffer. Leave the sample on ice for 30 minutes, and vortex every five minutes.Centrifuge the lysate at 14, 000 g, for 10 minutes at four degrees celsius, to pellet the detergent and soluble materials. Then, transfer the supernatant to a new microcentrifuge tube. Save 50 microliters of this lysate, and add loading buffer.Subsequently, denature the cells at 95 degrees celsius, in a dry bath. This is the input fraction, containing both biotinylated endocytosed proteins, as well as nonbiotinylated proteins. Using a cut pipette tip, add 150 microliters of the streptavidin agarose slurry to the lysate, and incubate it at four degrees celsius for three hours, on a shaker.After three hours, pellet the streptavidin agarose beads by centrifugation, at 1, 500 g for 30 seconds, at four degrees celsius. Resuspend the beads in one milliliter of wash buffer, and rock them for three minutes at four degrees celsius. Pellet the beads, and discard the supernatant.Repeat this process four more times to minimize the non-specific binding of nonbiotinylated cytosolic proteins. Then, pellet the beads by centrifugation at 1, 500 g for 30 seconds, at four degrees celsius. Discard the overlying wash buffer, and add 50 microliters of one x loading buffer.Release biotin and streptavidin from the beads, by denaturing at 95 degrees celsius. This fraction should contain internalized cell surface proteins only. Then, separate the input, cell surface, and unbound fractions, by SDS-PAGE, and analyze by Western blotting.Shown here, are the cell surface, intracellular, and total fractions from the untreated astrocytes, and astrocytes treated with 24 nanomolar laminin, probed for Aquaporin-4, and beta-dystroglycan. This histogram illustrates the differences in the cell surface levels of Aquaporin-4, in the untreated and laminin treated astrocytes, normalized against beta-dytroglycan. In this figure, the internalized protein fractions collected at zero, 15, and 30 minutes, were probed for Aquaporin-4.And this histogram, summarizes the internalization of Aquaporin-4, for four independent experiments, normalized against values for the zero minute time point. The asterisk indicates a statistically significant increase in the amount of Aquaporin-4, endocytosed between 15 and 30 minutes. When glutathione is used to break the disulfide bond in the cleavable biotin analog, the contribution of cell surface Aquaporin-4 to the biotinylated fraction, is sharply reduced.Resulting in a clear difference between the zero minute, and 15 minute samples. When the step is omitted, the signal originating from the cell surface pool, renders the change between the two time points undetectable. Following this procedure, other methods like immunofluorescence and TIRF Microscopy could be applied to answer additional questions, including physical trafficking of the cargo.

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Research

JoVE Journal

Neuroscience

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins.  Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein.  The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

This video demonstrates the isolation of cell-surface and intracellular proteins from an astrocyte culture. The astrocyte culture is placed on ice to inhibit endocytosis, and then the cell-surface proteins are labeled with a biotinylation reagent. The cells are lysed to release the biotinylated cell-surface proteins and non-biotinylated intracellular proteins. Finally, streptavidin-coated beads are used to separate the biotinylated cell-surface proteins from the intracellular proteins.

Isolation of Cell-Surface and Intracellular Proteins from an Astrocyte Culture

This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

Assessing the Internalization of  Target Surface Proteins Using a Biotin Derivative

Source: Creighton, B. A., et al. Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes. J. Vis. Exp. (2019)This video demonstrates the visualization of intracellular cargo transport in cultured astrocytes using a fluorescent acidotropic probe that selectively labels acidic endolysosomal vesicles. Time-lapse imaging and Z-stack acquisition with a confocal microscope allow tracking of fluorescently labeled cargo throughout the three-dimensional cell volume.

Visualizing Intracellular Transport of Cargo in Cultured Astrocytes

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

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

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

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Learn cell-surface and pulse-chase biotinylation methods to study Aquaporin-4 expression and endocytic rates in primary astrocyte cultures. These techniques reveal protein dynamics at the plasma membrane.

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

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