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

Summary

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.

Abstract

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.

Introduction

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 pathways^1,2,3,4,5. 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 c....

Protocol

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).

1. Using the method outlined in Noel et al.¹², prepare cultures of cortical astrocytes approximately 2 weeks in advance of the assay, and gr....

Results

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.......

Discussion

Modifications:
As these methods were designed for use with adherent cells, we have specified the use of PBS containing 100 mg/L MgCl₂∙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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium chloride (NH4Cl)	Fisher Scientific	A661-500
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A9647-50
Bromophenol blue	Bio-Rad	#1610404
cOmplete protease inhibitor cocktail	Sigma-Aldrich	11697498001
Disodium Ethylenediaminetetraacetate dihydrate (EDTA)	Bio-Rad	#1610729
Dithiothreitol (DTT)	Bio-Rad	#1610611
Dulbecco's Modified Eagle Medium (DMEM)	Gibco/Thermo Fisher Scientific	11960-044
EZ-Link Sulfo-NHS-LC-Biotin	Thermo Fisher Scientific	#21335
EZ-Link Sulfo-NHS-SS-Biotin	Thermo Fisher Scientific	#21331
Fetal Bovine Serum (FBS)	Gibco/Thermo Fisher Scientific	16000-044
Glycerol	Fisher Scientific	BP229-1
Glycine	Sigma-Aldrich	G8898
Iodoacetamide	Bio-Rad	#163-2109
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane	Sigma-Aldrich	L2020	Thaw on ice.
L-glutamine	Gibco/Thermo Fisher Scientific	25030-081
Mouse monoclonal anti-β-actin antibody (AC-15)	Sigma-Aldrich	A5441
Mouse monoclonal anti-β-dystroglycan antibody (43DAG1/8D5)	Leica Biosystems	B-DG-CE
Penicillin/streptomycin	Gibco/Thermo Fisher Scientific	15140-122
Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L)	Jackson ImmunoResearch Laboratories	715-035-150
Peroxidase AffiniPure Goat anti-Rabbit IgG (H+L)	Jackson ImmunoResearch Laboratories	111-035-045
Phosphate-Buffered Saline (PBS)	Gibco/Thermo Fisher Scientific	10010-023
Reduced glutathione	Sigma-Aldrich	G6529
Sodium chloride (NaCl)	Fisher Scientific	S271-500
Sodium Dodecyl Sulfate (SDS)	Sigma-Aldrich	862010
Sodium hydroxide (NaOH)	Fisher Scientific	S318-100
Streptavidin agarose resin	Thermo Fisher Scientific	#20347
Rabbit polyclonal anti-AQP4 antibody	Alomone	AQP-004
Tris base (Trizma base)	Fisher Scientific	BP152-1
Tris-HCl	Fisher Scientific	BP153-1
Triton X-100	Fisher Scientific	BP151-500