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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta+1+inhibits+epidermal+growth+factor+receptor+endocytosis+and+down-regulation+in+cultured+fetal+rat+hepatocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+internalization+of+fluorescent+gonadotropin-releasing+hormone+by+pituitary+gonadotropes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+uptake+and+release+by+reticulocytes+treated+with+proteolytic+enzymes+and+neuraminidase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+regulated+membrane+expression+of+aquaporin+1+and+aquaporin+2+water+channels+in+stably+transfected+LLC-PK1+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+family:+endocytosis+and+signal+transduction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possible+identity+of+vitamin+H+with+biotin+and+coenzyme+R.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation:+an+alternative+to+radioiodination+for+the+identification+of+cell+surface+antigens+in+immunoprecipitates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin+1.+the+use+of+[14-C]biotin+for+kinetic+studies+and+for+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin,+a+high+affinity+biotin-binding+protein,+as+a+tool+and+subject+of+biological+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pHluorin-tagged+receptor+insertion+to+the+plasma+membrane+in+primary+cultured+mouse+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+clathrin-mediated+endocytosis+of+G+protein-coupled+receptors+at+single-event+resolution+via+TIRF+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependence+of+laminin-mediated+clustering+of+lipid+rafts+and+the+dystrophin+complex+in+astrocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+trafficking+and+surface+expression+of+the+AQP4+water+channel+are+coordinated+by+serial+and+regulated+interactions+with+different+clathrin-adaptor+complexes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin-4+Cell-Surface+Expression+and+Turnover+Are+Regulated+by+Dystroglycan,+Dynamin,+and+the+Extracellular+Matrix+in+Astrocytes.">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 border="0" cellpadding="0" cellspacing="0" width="773">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

determining-cell-surface-expression-endocytic-rate-proteins-primary

Research

JoVE Journal

Neuroscience

9.4K Views.  University of British Columbia. 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.

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

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

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 ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

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

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

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.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...