Methods in cell biology, volume 132

Section I includes chapters on GPCR trafficking in lipid rafts and cilia, imaging endogenous receptor in neurons, single molecule imaging of GPCRs, and a comprehensive analysis of GPCRs

Philadelphia, USA &

Institut Curie, Paris, France

Edited by

Arun K Shukla

Department of Biological Sciences and Bioengineering,

Indian Institute of Technology, Kanpur, India

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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Notices

Knowledge and best practice in this field are constantly changing As new research andexperience broaden our understanding, changes in research methods, professionalpractices, or medical treatment may become necessary

Practitioners and researchers must always rely on their own experience and knowledge inevaluating and using any information, methods, compounds, or experiments describedherein In using such information or methods they should be mindful of their own safetyand the safety of others, including parties for whom they have a professional responsibility

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ISBN: 978-0-12-803595-5

ISSN: 0091-679X

For information on all Academic Press publications

visit our website athttp://store.elsevier.com

Agnes M Acevedo Canabal

Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,

San Juan, PR, USA; Department of Anatomy and Neurobiology, School of

Medicine, University of Puerto Rico, San Juan, PR, USA

Laboratory of Neural Systems (SisNE), Department of Physics, Faculdade de

Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo,

Ribeira˜o Preto, Brazil

Mohammed Akli Ayoub

Biologie et Bioinformatique des Syste`mes de Signalisation, Institut National de la

Recherche Agronomique, UMR85, Unite´ Physiologie de la Reproduction et des

Comportements; CNRS, UMR7247, Nouzilly, France; LE STUDIUMÒLoire Valley

Institute for Advanced Studies, Orle´ans, France

R Bar-Shavit

Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,

Jerusalem, Israel

Damian Bartuzi

Department of Synthesis and Chemical Technology of Pharmaceutical

Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of

Medical Analytics, Medical University of Lublin, Lublin, Poland

Maik Behrens

Department of Molecular Genetics, German Institute of Human Nutrition

Potsdam-Rehbruecke, Nuthetal, Germany

xiii

Colleen A Flanagan

School of Physiology and Medical Research Council Receptor Biology Research

Unit, Faculty of Health Sciences, University of the Witwatersrand, Wits Parktown,

Johannesburg, South Africa

Alexandre Gidon

Molecular Mechanisms of Mycobacterial Infection, Center for Molecular

Inflammation Research, Norwegian University of Science and Technology,

Trondheim, Norway

Claudia Gonza´lez-Espinosa

Departamento de Farmacobiologı´a, Centro de Investigacio´n y de Estudios

Avanzados del IPN, Me´xico D.F., Mexico

Department of Biology, Indiana University-Purdue University Indianapolis,

Indianapolis, IN, USA

Institute of Reproductive and Developmental Biology, Imperial College London,

London, UK; Institute of Medical and Biomedical Education, St George’s

University of London, London, UK

Pedro A Jose

Division of Renal Diseases & Hypertension, Department of Medicine, The George

Washington University School of Medicine and Health Sciences, WA, USA

Manali Joshi

Savitribai Phule Pune University, Pune, India

Agnieszka A Kaczor

Department of Synthesis and Chemical Technology of Pharmaceutical

Substances with Computer Modelling Lab, Faculty of Pharmacy with Division ofMedical Analytics, Medical University of Lublin, Lublin, Poland; School ofPharmacy, University of Eastern Finland, Kuopio, Finland

Dariusz Matosiuk

Department of Synthesis and Chemical Technology of Pharmaceutical

Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of

Medical Analytics, Medical University of Lublin, Lublin, Poland

Jeremy C McIntyre

Department of Neuroscience, University of Florida, Gainesville, FL, USA; Center

for Smell and Taste, University of Florida, Gainesville, FL, USA

Masha Y Niv

Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith

Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot,

Israel; Fritz Haber Center for Molecular Dynamics, The Hebrew University,

Jerusalem, Israel

Carlos Nogueras-Ortiz

Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,

San Juan, PR, USA

Melanie Philipp

Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany

Cristina Roman-Vendrell

Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,

San Juan, PR, USA; Department of Physiology, School of Medicine, University of

Puerto Rico, San Juan, PR, USA

Ewelina Rutkowska

Department of Biopharmacy, Faculty of Pharmacy with Division of Medical

Analytics, Medical University of Lublin, Lublin, Poland

Jana Selent

Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu

Fabra, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain

Durba Sengupta

CSIR-National Chemical Laboratory, Pune, India

Ying Shi

Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang

University, Hangzhou, Zhejiang, China

Fabio Marques Simoes de Souza

Center for Mathematics, Computation and Cognition, Federal University of ABC,

Sa˜o Bernardo do Campo, Brazil

Michal Slutzki

Institute of Biochemistry, Food Science and Nutrition, The Robert H SmithFaculty of Agriculture, Food and Environment, The Hebrew University, Rehovot,Israel

Teresa Casar Tena

Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany

Van Anthony M Villar

Division of Renal Diseases & Hypertension, Department of Medicine, The GeorgeWashington University School of Medicine and Health Sciences, WA, USA

Yaping Zhang

Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang

University, Hangzhou, Zhejiang, China

Xiaoxu Zheng

Division of Renal Diseases & Hypertension, Department of Medicine, The George

Washington University School of Medicine and Health Sciences, WA, USA

Cynthia Zhou

Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC,

Canada

Naiming Zhou

Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang

University, Hangzhou, Zhejiang, China

G proteinecoupled receptors (GPCRs) also referred as seven transmembrane

receptors (7TMRs) lie at the heart of almost every physiological and

pathophysio-logical process in our body These receptors bind to and get activated by a wide

range of ligands ranging from small molecules, hormones, peptides, proteins to

lipids The overall activation and signal transduction mechanisms of GPCRs are

highly conserved where binding of an agonist results in a conformational change

in the receptor followed by activation of heterotrimeric G proteins and subsequent

generation of second messengers and downstream signaling Downregulation of

GPCRs is also primarily a conserved process where activated receptors are

phosphorylated by GRKs (GPCR kinases) followed by binding of beta arrestins

which leads to receptor desensitization and internalization GPCRs are targeted by

about one-third of the currently prescribed drugs which include angiotensin blockers

for hypertension, beta-blockers for heart failure, antihistamines for allergy

manage-ment, and opioid agonists as analgesic medication

In this volume of Methods in Cell Biology, we cover multiple aspects of GPCR

signaling, trafficking, regulation, and cellular assays in a form of either an

over-view or as step-by-step protocol This is an effort to bring together different

domains of GPCR pharmacology and signaling on to a common platform and

high-light the incredibly versatile nature and diverse functional manifestation of

GPCRs Section I includes chapters on GPCR trafficking in lipid rafts and cilia,

imaging endogenous receptor in neurons, single molecule imaging of GPCRs,

and a comprehensive analysis of GPCRs in adipose tissue In Section II, we cover

topics ranging from GPCR signaling from endosomes, olfactory receptor signal

transduction, studies of a specialized GPCR smoothened in zebra fish model,

and the outcome of GPCR signaling in cytoskeletal dynamics In recent years, a

key focus area in GPCR biology has been the development of novel and more

sen-sitive cellular assays to investigate GPCR expression, signaling, and

downregula-tion Section III of this volume is focused on GPCR assays which include classical

radioligand binding, label-free, biosensor and fluorescenceebased approaches to

study GPCR trafficking and signaling, and TANGO assay for measuring

GPCR-beta-arrestin interaction Finally, Section IV consists of chapters on structural

and computational aspects of protease-activated receptors, bitter taste receptors,

and GPCR dimerization

I would like to thank all the authors who have contributed to this focused volume

despite their busy schedule I also express my sincere gratitude to the journal

edito-rial staff and production team for a wonderful job in putting this volume together in a

timely fashion With this brief background, on behalf of the entire Methods in Cell

xxi

Biology Team, I present to you this volume entitled “G ProteineCoupled Receptors:Signaling, Trafficking, and Regulation.” I sincerely hope that you enjoy the topicscovered in this issue and please feel free to share your feedback with us.

Arun K ShuklaIndian Institute of Technology, Kanpur, India

Localization and signaling

Van Anthony M Villar 1 , Santiago Cuevas, Xiaoxu Zheng, Pedro A Jose 1

Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington

University School of Medicine and Health Sciences, WA, USA

1 Corresponding authors: E-mail: vvillar@gwu.edu; pjose@mfa.gwu.edu

CHAPTER OUTLINE

Introduction 4

1 Localization of GPCRs in Lipid Rafts 6

1.1 Isolation of Lipid Rafts 7

1.1.1 Detergent-free method 7

1.1.2 Detergent-based method 9

1.1.3 Immunoblotting and data interpretation 10

1.2 Localization of GPCRs in Lipid Rafts 11

1.2.1 Cells in suspension 13

1.2.2 Adherent cells 14

2 GPCR Signaling in Lipid Rafts 15

2.1 Perturbation of Raft Stability 15

2.2 Changing the Cholesterol Content 16

2.3 Fluorescence Imaging 16

References 18

Abstract

The understanding of how biological membranes are organized and how they function

has evolved Instead of just serving as a medium in which certain proteins are found,

portions of the lipid bilayer have been demonstrated to form specialized platforms that

foster the assembly of signaling complexes by providing a microenvironment that is

conducive for effective proteineprotein interactions G protein-coupled receptors

(GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key

enzymes such as kinases and phosphatases, trafficking proteins, and secondary

messen-gers, preferentially partition to these highly organized cell membrane microdomains,

called lipid rafts As such, lipid rafts are crucial for the trafficking and signaling of

GPCRs The study of GPCR biology in the context of lipid rafts involves the localization

of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and

Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.11.008

© 2016 Elsevier Inc All rights reserved. 3

the evaluation of the biological functions of the GPCR in appropriate cell lines The lack

of standardized methodology to study lipid rafts, in general, and of the workings ofGPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods havehampered the complete understanding of the underlying molecular mechanisms Newermethodologies that allow the study of GPCRs in their native form are needed The use ofcomplementary approaches that produce mutually supportive results appear to be the bestway for drawing conclusions with regards to the distribution and activity of GPCRs inlipid rafts

INTRODUCTION

Lipid Raft Microdomains The plasma membrane is a semipermeable, biologicalmembrane that demarcates the intracellular milieu from the extracellular environ-ment Amphipathic lipids, such as phospholipids and sphingolipids, are the buildingblocks of these bilipid membranes because of their aggregative properties, i.e., theirhydrophobic tails associate together, while their hydrophilic heads interact with bothextra- and intracellular aqueous environments (Sonnino & Prinetti, 2013) Thefluidity of the fatty acyl groups of phospholipids at 37
C enables the membranes

to act as a medium in which dissolved membrane proteins are afforded ample lateralmobility, especially in response to environmental cues Since the first description of

an “organization of the lipid components of membranes into domains” (Karnovsky

et al., 1982) and the elaboration of the “lipid raft hypothesis” by Simons and vanMeer (van Meer & Simons, 1988; Simons & Ikonen, 1997; Simons & van Meer

1998), the existence of lipid rafts is now established

Lipid rafts are tightly packed, highly organized plasma membrane microdomainsthat are enriched in phospholipids, glycosphingolipids, and cholesterol and serve as

a platform for the organization and dynamic interaction of biomolecules involved invarious biological processes (Figure 1) The cholesterol bestows a semblance ofrigidity and order by intertwining into the hydrophobic gaps between the phospho-lipid acyl chains Certain structural proteins abound in lipid rafts to serve as scaffold

or anchor for other proteins, including caveolins (Head, Patel, & Insel, 2014; Quest,Leyton, & Pa´rraga, 2004; Yu, Villar, & Jose, 2013; Yu et al., 2004), flotillins(Rajendran, Le Lay, & Illges, 2007; Yu et al., 2004) and tetraspanins (Hemler,

2005), and glycosylphosphatidylinositol-linked (GPI-linked) proteins The spatialconcentration and organization of specific sets of membrane proteins allow greaterefficiency and specificity of signal transduction by facilitating proteineproteininteractions and by preventing crosstalk between competing pathways Thenonhomogeneous lateral distribution of membrane components helps explain thedifferences in composition between apical and basolateral membrane domains ofpolarized epithelial cells (Sonnino & Prinetti, 2013)

The best characterized lipid raft microdomains are the caveolae, which were firstdescribed by Palade and Yamada in the 1950s (Palade, 1953; Yamada, 1955) Theseare small (60e80 nm) invaginations of the plasma membrane formed by thepolymerization of caveolins with cholesterol (Parton & del Pozo, 2013) Caveolae

have been implicated in a variety of cellular processes, including signal

transduc-tion, endocytosis, transcytosis, and cholesterol trafficking (Barnett-Norris, Lynch, &

Reggio, 2005) Lipid rafts accumulate in the apical plasma membrane in polarized

epithelial cells and in axonal membranes in neurons Basolateral and dendritic

membranes contain lipid rafts but in more limited quantities (Simons & Ikonen,

1997) Interestingly, caveolae are found mostly at the basolateral membrane that

faces the blood supply and is more active during signal transduction (Simons &

Toomre, 2000) Lipid rafts are mostly found at the plasma membrane; however,

they may also be found in intracellular membranes involved in the biosynthetic

and endocytic pathways Lipid raft microdomains play a crucial role in cellular

pro-cesses such as membrane sorting, receptor trafficking, signal transduction, and cell

adhesion

GPCR Signaling and Trafficking G protein-coupled receptors (GPCRs)

constitute the largest superfamily of seven transmembrane proteins that respond

to a myriad of environmental stimuli that are transduced intracellularly as

meaning-ful signals through secondary messengers Agonist stimulation of a GPCR leads to a

conformational change that promotes the exchange of GDP for GTP on the Ga

sub-unit of the G protein, resulting in the uncoupling of the G protein from the GPCR and

the dissociation of Gaand Gbgsubunits The Gasubunit either activates or inhibits

intracellular signaling pathways depending on the receptor subtype, while the Gbg

subunit recruits G protein-coupled receptor kinases which selectively phosphorylate

serine and threonine residues localized within the third intracellular loop and

carboxyl-terminal tail domains of the receptor to promote the binding of cytosolic

cofactor proteins called arrestins (Lefkowitz, 1998) The b-arrestins play a pivotal

role in the uncoupling process and in the sequestration and internalization of GPCRs

FIGURE 1 A Lipid Raft Membrane Microdomain.

Lipid rafts are highly organized plasma membrane microdomains enriched in phospholipids,

glycosphingolipids, and cholesterol, and serve as matrix for receptors, such as G

protein-coupled receptors (GPCRs), and other signaling molecules (See color plate)

Van Anthony M Villar, MD, PhD.

through a dynamin-dependent, clathrin-mediated endocytosis Once internalized,the GPCRs, in vesicles termed as early endosomes, are sorted by sorting nexinsand follow divergent pathways (Worby & Dixon, 2002) The receptors are sortedinto recycling endosomes for their return to the cell membrane (recycling andresensitization), accumulate in late endosomes which target the lysosomes for theirsubsequent degradation, or transported initially to the perinuclear endosomes (trans-Golgi network) and then to the late endosomes for eventual lysosomal degradation.Additional proteolytic mechanisms, such as proteasomes or cell-associated endo-peptidases, are also implicated in mediating the downregulation of certain GPCRs(von Zastrow, 2003).

The signal transduction that follows ligand occupation of the GPCR is highlyregulated to ensure the specificity of the cellular response, both temporally andspatially The signal transduction can be attenuated with relatively fast kineticsthrough a process called desensitization or through a much slower process of down-regulation following prolonged or repeated exposure to an agonist Desensitization,

or the waning of a receptor’s responsiveness to agonist with time, is an inherentmolecular “feedback” mechanism that prevents receptor overstimulation and helps

in creating an integrated and meaningful signal by filtering out information fromweaker GPCR-mediated signals (Ferguson, 2001)

It is accomplished through two complementary mechanisms, i.e., the functionaluncoupling of GPCRs from their cognate G proteins, which occurs without anydetectable change in the number of cell surface receptors, and GPCR phosphoryla-tion, sequestration, and internalization/endocytosis GPCR resensitization protectsthe cells from prolonged desensitization and is carried out via dephosphorylation

by phosphatases as the GPCR traffics through the endosomal pathway GPCR ity is the net result of a coordinated balance between receptor desensitization andresensitization

activ-It is now established that lipid rafts serve as dynamic platforms for GPCRs andpertinent signaling molecules such as G proteins, enzymes, and adaptors (Barnett-Norris et al., 2005; Lingwood & Simons, 2010) However, understanding themolecular mechanisms involved has been hampered by the lack of standardizedmethodology to study lipid rafts, in general, and of the workings of GPCRs in lipidrafts, in particular Moreover, the minute size of lipid rafts has made lipid raftsdifficult to resolve by standard light microscopy, unless the lipid raft componentsare cross-linked with antibodies or lectins (Simons & Toomre, 2000) Studyinghow GPCR works in lipid rafts may be accomplished by determining if theGPCR of interest localizes to the lipid rafts and by evaluating if GPCR signalingand activity are lost when lipid rafts are disrupted

Several techniques are available for the detection and localization of GPCRs in lipidraft microdomains in cells The most commonly employed approach utilizes cell

fractionation procedures that break the cells apart and destroy cell morphology

before GPCR analysis using biochemical or immunological assays A

complemen-tary biophysical approach involves the visualization of GPCRs in intact cell

membranes

Lipid rafts are characterized by their relative insolubility in nonionic detergents at

4
C and light buoyant density on sucrose gradient (Schnitzer, McIntosh, Dvorak,

Liu, & Oh, 1995) The isolation of lipid rafts can be performed using either

detergent-based or detergent-free methods (Yu et al., 2013), with the latter generating

a greater fraction of inner leaflet membrane rafts and producing more replicable

results (Pike, 2004).Schnitzer et al (1995) employed a detergent-free method to

isolate lipid rafts using cationic colloidal silica particles, which is appropriate for

non-cell culture studies Lipid rafts may be extracted from total cell membranes

(Song et al., 1996) or just from surface plasma membranes (Smart, Ying, Mineo, &

Anderson, 1995) Detergent insolubility results from the segregation of

membrane-associated proteins into the lipid rafts, which are abundant in cholesterol and

glycosphingolipids Nonionic detergents, such as Triton X-100, b-octyl glucoside,

CHAPS, deoxycholate, Lubrol WX, Lubrol PX, Brij 58, Brij 96, and Brij 98, have

been used to prepare lipid raft fractions (Macdonald & Pike, 2005), resulting in

varying yields of proteins Samples obtained by detergent-based methods are termed

detergent-resistant membranes or detergent-insoluble fractions Different detergents

may yield different lipid raft components because of the varying degrees of

resis-tance by the proteins to extraction using different reagents The methods detailed

below are based onYu et al (2013)

1.1.1 Detergent-free method

Materials

2-N-morpholino ethanesulfonic acid (Mes), 250 mM, pH¼ 6.8

Mes-buffered solution (MBS), 25 mM Mesþ 150 mM NaCl

Sodium citrate, 500 mM, pHw 11 (add protease inhibitors)

Sucrose, 5%, 35%, and 80% in MBS solution (add protease inhibitors)

Methyl-b-cyclodextrin (b-MCD), 2% dissolved in cell culture media

Cholesterolþ b-MCD (Sigma catalog #C4951), dissolved in cell culture

media

1X PBS, for washing

1 Cell culture and cell pellet collection To obtain sufficient amounts of lipid raft

fraction, cells should be grown in 150-mm dishes until almost confluent using

the appropriate media at 37
C with 95% air and 5% CO2 Separate dishes of

cells should also be treated for cholesterol depletion and repletion as

experi-mental controls (Figure 2) Cholesterol depletion to disrupt the lipid rafts is

commonly performed by pretreatment with b-MCD for 1 h at 37
C.

Methyl-a-cyclodextrin (a-MCD) may be used as control for b-MCD (Vial &Evans, 2005) Cholesterol repletion is performed by pretreating with choles-terol/b-MCD solution for 1 h at 37
C Cholestane-3,5,6-triol, an inactive

analog of cholesterol, may be used as control for the use of exogenous

cholesterol (Murtazina, Kovbasnjuk, Donowitz, & Li, 2006) To determine theeffect of agonist or antagonist treatment, cells should be serum-starved for atleast 1 h prior to treatment to achieve “basal” conditions prior to treatment.Additional controls, such as the use of the drug vehicle, should be concomi-tantly performed

1.1 Wash cells with cold PBS and scrape the cells using a rubber-tipped cell

scraper

1.2 Transfer cell suspension into 15-mL tube and spin at 2000 g for 5 min

1.3 Decant the supernatant to obtain the cell pellet.

2 Cell homogenate preparation All steps are carried out at 4
C.

2.1 To the cell pellet, add 1.5 mL 500 mM sodium carbonate and vortex 2.2 Homogenize the cell suspension by sonication using five 20-s bursts on ice 2.3 Add 1.5 mL of 80% sucrose and mix by vortex and sonication (three 20-s

bursts) on ice Protein concentration may be determined at this time using aBCA kit

3 Sucrose gradient ultracentrifugation Prepare 5%, 35%, and 80% sucrose

solutions in MBS solution The use of MBS solution with pH close to 7.0 may

be advantageous for most proteins

3.1 Place 3 mL of cell homogenates into the bottom of precooled 12-mL

4 Lipid raft fraction preparation A light-scattering band that is enriched with

caveolae/lipid rafts can be observed between the 5% and 35% sucrose gradientsand corresponds to the fourth fraction

4.1 Carefully aspirate 12 1-mL fractions from the top of the tube and transfer

into prelabeled 1.5 microcentrifuge tubes

FIGURE 2 Comparison groups for GPCR localization in lipid rafts.

4.2 Prepare 0.5 mL of each fraction by adding 0.1 mL 6X sample buffer, vortex,

and boil for 5 min before use for immunoblotting These samples can be

stored at20
C, while the rest of the fractions without the 6X sample

buffer can be stored at80
C.

1.1.2 Detergent-based method

Materials

50% Optiprep Stock solution (45 mL of 60% Optiprepþ 9 mL of Optiprep

diluent)

MBSTS buffer (MBSþ 0.5% Triton X-100 þ protease inhibitors in 10% sucrose)

Sucrose solutions (Table 1):

1 Cell culture and cell pellet preparation The same as with the detergent-free

method

2 Cell extract preparation.

2.1 Add 0.3 mL ice-cold MBSTS to cell pellet and push through a 25G

needle 10

2.2 Adjust cell extract (w0.4 mL; cell pellet volume is w0.1 mL) to 40%

Optiprep by adding 0.8 mL of cold 60% Optiprep and vortex Determine

protein concentration using a BCA kit

3 Optiprep gradient ultracentrifugation.

3.1 Place 1 mL of the cell extract into the bottom of precooled 5-mL

ultra-centrifuge tubes

3.2 Overlay with 1 mL each of 30%, 25%, 20%, and 0% Optiprep solutions in

MBSTS buffer

3.3 Secure each tube in a Beckman SW 50.1 bucket and spin at 175,000 g

(42,000 rpm) at 4
C for 4 h Other rotors may be used, such as the SW 55

(170,000 g for 4 h) or TLS55 (250,000 g for 2.5 h)

4 Lipid raft fraction preparation.

4.1 Carefully aspirate ten 0.5-mL fractions from the top of the tube and transfer

into prelabeled 1.5 microcentrifuge tubes

4.2 Prepare 0.25 mL of each fraction by adding 0.5 mL 6X sample buffer,

vortex, and boil for 5 min before use for immunoblotting These samples

can be stored at20
C, while the rest of the fractions without the 6X

sample buffer can be stored at80
C.

Table 1 Preparation of Optiprep Gradient Solutions

1.1.3 Immunoblotting and data interpretation

Western blot is the most commonly used method to determine the lipid raft tion of proteins, such as GPCRs Antibody specificity is crucial for the identification

distribu-of the GPCR distribu-of interest The lipid raft proteins are found in the more buoyantfractions (top 5e6 fractions); however, their distribution among these fractions isnot uniform Immunoblotting for lipid raft markers may help in determining thefractions where the lipid rafts are most abundant Caveolin-1 is the most commonlyused protein marker for lipid rafts, specifically for caveolae (Insel et al., 2005;Lingwood & Simons, 2010) There are several other markers for lipid rafts, such

as flotillin-1, CD55, alkaline phosphatase, and pore-forming toxins, such as choleratoxin subunit B (CTxB), equinatoxin II, perfringolysin (Foster, De Hoog, & Mann,2003; Salzer & Prohaska, 2001; Skocaj et al., 2013) Flotillin-1 has been used as alipid raft marker protein in cells that do not contain caveolae, i.e., blood cells(Salzer & Prohaska, 2001), neural cells (Huang et al., 2007), and rat renal proximaltubule cells (Breton, Lisanti, Tyszkowski, McLaughlin, & Brown, 1998; Riquier, Lee,

& McDonough, 2009) and human embryonic kidney (HEK)-293 cells (Yu et al.,

2004) There is species specificity because human renal proximal tubule cellsexpress caveolin-1 (Gildea et al., 2009), while HEK-293 cells express caveolin-2.These markers may also be used to indicate the integrity of lipid rafts in cholesteroldepletion or repletion experiments In general, these markers should be distributed inthe more buoyant fractions and should redistribute into the less buoyant fractions(fractions 7e12) after cholesterol depletion with b-MCD (Figure 3) Cholesterolrepletion reconstitutes the lipid rafts and thus, these markers should be observed inthe more buoyant fractions

FIGURE 3 Lipid Raft Distribution of Caveolin-1 and D 1 R.

Lipid raft and non-lipid raft fractions from human renal proximal tubule cells treated withb-MCD, a cholesterol-depleting and lipid raft-disrupting agent, were prepared by detergent-free method and sucrose gradient ultracentrifugation The distribution of caveolin-1, a lipidraft marker, and the dopamine D1receptor (D1R), a GPCR, is shown in the immunoblots

Images are courtesy of Peiying Yu, MD.

1.2 LOCALIZATION OF GPCRs IN LIPID RAFTS

Another way to demonstrate the distribution of GPCRs in lipid rafts is by visualizing

them in intact cells, living or fixed, and tissues There are now commercially

avail-able kits that have been developed for labeling the lipid rafts using the CTxB that is

tagged with fluorophores (Figure 4) CTxB binds to the pentasaccharide chain of

ganglioside GM1, which selectively partitions into lipid rafts For visualizing lipid

rafts, cells are labeled with CTxB tagged with Alexa FluorÒ488, Alexa FluorÒ

FIGURE 4 Colocalization of the D 1 dopamine receptor (D 1 R) in Lipid Rafts of Human Renal

Proximal Tubule Cells.

Human renal proximal tubule cells were grown on a poly-L-Lysine-coated cover slip to 50%

confluence and serum-starved for 1 h to determine the basal distribution of D1R prior to

fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100 The lipid

rafts were labeled using cholera toxin subunit B (CTxB) tagged with Alexa FluorÒ555

(Molecular Probes), while the endogenous D1R was immunostained using a proprietary

rabbit-anti-D1R antibody and a donkey anti-rabbit secondary antibody tagged with Alexa

FluorÒ488 (Molecular Probes) DAPI was used to visualize the nucleus At the basal state,

most of the D1R were found intracellularly, just below the inner leaflet of the plasma

membrane, although some colocalized with the lipid rafts (yellow areas pointed at by arrows)

The raw images were captured via laser scanning confocal microscope using separate

channels and the composite image was obtained using Zen 2011 software 630X

magnification, scale bar¼ 10 mm (See color plate)

Van Anthony M Villar, MD, PhD.

555, or Alexa FluorÒ647 before cross-linking with an anti-CTxB to maintain the

in situ protein distribution To demonstrate the lipid raft distribution of GPCRs, alization experiments may be performed via laser scanning confocal microscopy bylabeling the lipid rafts using CTxB and immunostaining the GPCR of interest usingspecific antibodies on the same cell CTxB labeling may also be used to demonstratelipid raft endocytosis upon agonist stimulation in live cells (Qi, Mullen, Baker, &Holl, 2010) and cultured explants (Hansen et al., 2005) The c-subunit of cytolethaldistending toxin (cdt) may also be utilized for lipid raft colocalization experiments(the protocol is detailed inBoesze-Battaglia, 2006) Other pore-forming toxins, be-sides CTxB, used to visualize lipid rafts include equinatoxin II which binds dispersedsphingomyelin, lysenin which binds clustered sphingomyelin, perfringolysin O whichbinds to cholesterol, and ostreolysin which binds to the combination of sphingomyelinand cholesterol (Makino et al., 2015; Skocaj et al., 2013)

coloc-An alternative to using CTxB, cdt, and other pore-forming toxins is to useantibodies that specifically target the lipid raft protein markers, such ascaveolin-1, caveolin-3, and flotillin-1 Conversely, transferrin receptors, CD71,and geranylated proteins are non-lipid raft markers (Boesze-Battaglia, 2006;Magee, Adler, & Parmryd, 2005) The ganglioside GM1may be labeled with singlequantum dots to measure the lateral mobility and extent of movement of the lipidrafts (Chang & Rosenthal, 2012) Recently, GPI-anchored proteins that segregateinto lipid rafts have been visualized using a novel method called enzyme-mediatedactivation of radical sources (Miyagawa-Yamaguchi, Kotani, & Honke, 2015).Probes that target the lipid content of lipid rafts have also been used to visualizethese membrane microdomains Laurdan (6-dodecanoyl-2-(dimethylamino)-naphthalene) and C-laurdan (6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]-naphthalene), which are membrane probes that are sensitive to membranepolarity, allow the observation of lipid rafts via two-photon microscopy (Gaus,Zech, & Harder, 2006; Kim et al., 2007, 2008) A fluorophore-tagged domainD4 of perfringolysin O, a cholesterol-binding cytolysin produced by Clostridiumperfringens, has been used as probe to study membrane cholesterol (Ohno-Iwashita et al., 2004)

Aside from confocal microscopy, other biophysical approaches may also beemployed to study labeled GPCRs and/or lipid rafts Single fluorophore trackingmicroscopy (Schu¨tz, Kada, Pastushenko, & Schindler, 2000) and fluorescencerecovery after photobleaching (Kenworthy, 2007) may be used to monitor lateraldiffusion of lipid raft-anchored GPCRs, while fluorescence lifetime imagingmicroscopyefluorescence resonance energy transfer (FLIM-FRET) (Kenworthy,Petranova & Edidin, 2000; Thaa, Herrmann, & Veit, 2010) may be used to deter-mine the proximity of GPCRs with other proteins of interest, or of lipid raft sizesdepending on membrane composition (de Almeida, Loura, Fedorov, & Prieto,

2005) Atomic force microscopy may be used to visualize the effects of detergentsolubilization of membranes during lipid raft studies (Garner, Smith, & Hooper,

2008) Lipid rafts can now be visualized using superresolution imaging belowthe 200 nm limit of conventional microscopes, e.g., including structured

illumination microscopy, stimulated emission depletion (STED) microscopy,

near-field scanning optical microscopy, photoactivated localization microscopy

(PALM), and stochastic optical reconstruction microscopy (dSTORM) (Owen &

Gaus, 2013; Tobin et al., 2014; Wu et al., 2013)

Materials

VybrantÒLipid Raft Labeling Kits (Catalog #V-34403, V-34404, or V-34405)

prepare fresh working solutions according to manufacturer’s instructions

Primary antibody against the GPCR of interest

Secondary antibody against the host of the primary antibody

10% bovine serum albumin (BSA) solution

4% Paraformaldehyde in PBS

Mounting medium (EMS catalog #17985) without 40

,6-diamidino-2-phenylindole (DAPI)

DAPI, a nuclear stain, 10 mM stock solution

Triton X-100, 20% stock solution in deionized water

1X PBS for washing

1.2.1 Cells in suspension

Colocalization of GPCRs with lipid rafts can now be accomplished with the

concom-itant use of CTxB and an antibody against the GPCR of interest on cells The cells can

be labeled in suspension and then mounted on glass slides for imaging, or the cells can

be grown and labeled on cover slips or in TranswellsÒcell culture inserts when cell

polarity is important to distinguish between apical versus basolateral membranes

1 Fluorescent labeling of cells.

1.1 Spin cells at 2000 g for 5 min and decant the medium

1.2 Resuspend the cells in cold medium, spin, and decant the medium.

1.3 Resuspend the cells in 2 mL of CTxBeAlexa FluorÒworking solution at

4
C for 10 min The primary antibody against the GPCR of interest may

be added to this working solution at 1:100 dilution The primary antibody

against the GPCR should be raised in mouse, goat, rat, or chicken but not in

rabbit when using the VybrantÒLipid Raft Labeling Kits Alternatively,

the primary antibody against the GPCR (especially if only a rabbit

anti-body is available) may be prelabeled with a Fluor other than the one used

for CTxB Directly labeling the primary antibody precludes the use of a

secondary antibody (in step 1.5)

1.4 Gently wash cells 3 with cold PBS Spin cells and decant wash buffer

1.5 Resuspend in 2 mL of the rabbit CTxB antibody working solution at 4
C

for 30 min The rabbit CTxB antibody cross-links it to the lipid raft

do-mains The secondary antibody against the primary antibody may be added

to this working solution at 1:100 dilution The secondary antibody should

be tagged with a Fluor other than the one used to label the CTxB

As counterstain, 300 nM DAPI may also be added to this working solution

1.6 Gently wash cells 3 with cold PBS Spin cells and decant wash buffer

2 Mounting and imaging.

2.1 (Optional) Fix cells with 4% paraformaldehyde at room temperature for

15 min Paraformaldehyde is a cross-linker fixative that preserves thearchitecture of the cell but may reduce the antigenicity of some cellcomponents and thus, requires an additional permeabilization step ifadditional intracellular proteins are needed to be visualized Fixation mayalso be achieved using organic solvents, such as alcohols and acetone, butthese remove lipids and precipitate the proteins and often disrupt the cellstructure

2.2 Mount live cells in cold PBS or fixed cells in mounting medium on glass

slide and cover with cover slip

2.3 Image the cells using a laser scanning confocal microscope The appropriate

filters should be used depending on the Alexa FluorÒdye that was used andwhether DAPI was used as a nuclear stain or not (Table 2)

1.2.2 Adherent cells

1 Cell culture on cover slips.

1.1 Grow cells on 12-mm cover slips placed in a 24-well tissue culture plate to

w50% confluence using complete cell culture medium at 37
C in 95% air

and 5% CO2 Cover slips coated with lysine, laminin, or collagen mayimprove cell attachment for cells that easily detach, such as HEK-293 cells

To determine the effect of agonist/antagonist treatment on GPCRtrafficking, cells should be serum-starved for at least 1 h prior to treatment

to achieve “basal” conditions prior to treatment Additional controls, such

as vehicle treatment, should be performed

1.2 Draw off the medium and wash cells with cold PBS Place the cell culture

plate on ice to stop further receptor endocytosis and endosomal trafficking

2 Fluorescent labeling, fixation, and permeabilization.

2.1 Add 0.3 mL of CTxBeAlexa FluorÒworking solution at 4
C for 10 min.

2.2 Draw off the solution and wash cells with cold PBS.

2.3 Fix cells with 0.3 mL of 4% paraformaldehyde at room temperature for

15 min

2.4 Wash cells with PBS Subsequent steps can be performed at room

temperature

Table 2 Fluorescence Spectra of CTxB Conjugates

CTxB Fluor Conjugate (Catalog #)

Maximum Absorption and Emission (nm)

Alexa FluorÒ488 (V-34403) 495/519

Alexa FluorÒ555 (V-34404) 555/565

Alexa FluorÒ594 (V-34405) 590/617

The maximum absorption and emission for DAPI are 358/461 nm.

2.5 Permeabilize the cells with 0.3 mL of 0.5% Triton X-100 in deionized water

for 10 min Permeabilization provides access to intracellular antigens

Triton X-100 can effectively solvate cellular membranes without

disturb-ing proteineprotein interactions Other detergents such as saponin,

Tween-20, or sodium dodecyl sulfate may also be used

2.6 Wash cells with PBS.

3 Immunostaining.

3.1 Add 0.3 mL of the primary antibody against the GPCR of interest dissolved

in 10% BSA (1:100e200 dilution) for 30e60 min

3.2 Wash cells 3X with PBS.

3.3 Add 0.3 mL of the secondary antibody (against the host of the primary

antibody used in step 3.1) in 10% BSA The secondary antibody should be

tagged with a Fluor other than the one used to label the CTxB As

coun-terstain, 300 nM DAPI may also be added to this working solution

3.4 Wash 2X with PBS and once with deionized water The use of deionized

water washes away the residual NaCl crystals from PBS

3.5 Mount cover slips using a mounting medium on glass slide Gently remove

excess mounting medium by aspiration Allow the mounting medium to

harden completely

3.6 Image the cells using a laser scanning confocal microscope The appropriate

filters should be used depending on the Alexa FluorÒdye that was used and

whether DAPI was used as a nuclear stain

There are many established protocols available that allow the study of GPCR activity

per se using commercially available kits or, less commonly, proprietary materials

Studying the activity of GPCRs in the context of their residency in lipid rafts often

requires additional steps that would disrupt the integrity of the lipid raft

microdo-main or dissociate the protein of interest from the rafts Most of the current strategies

to disrupt lipid raft involves either perturbation of the raft stability or modifying the

cholesterol content of the lipid rafts Most of these treatments are performed on cells

prior to agonist/antagonist treatment and functional assays, such as cAMP

produc-tion, sodium transport, and NADPH oxidase activity (Gildea et al., 2009; Han et al.,

2008; Yu et al., 2004, 2014)

Lipid rafts are dynamic assemblies of phospholipids and glycosphingolipids that

contain mostly saturated hydrocarbon chains which allow cholesterol to intercalate

between the fatty acyl chains The surrounding membrane has greater fluidity

because of the preponderance of phospholipids with unsaturated acyl groups The

addition of exogenous gangliosides (Webb, Hermida-Matsumoto, & Resh, 2000)

and polyunsaturated fatty acids (Simons et al., 1999), such as docosahexaenoic acid(Ravicci et al., 2013), in the growth medium results in a change in the lipid raftcomposition and the dissociation of proteins from the lipid raft Inhibition of thebiosynthesis of glycosphingolipids and sphingomyelins using the fungal metabolitefumonisin B1 (Lipardi, Nitsch, & Zurzolo, 2000; Nakai & Kamiguchi, 2002) mayalso perturb the integrity of lipid rafts Supplementation with 7-ketocholesterol,which differs from cholesterol by the additional ketone group that protrudes perpen-dicularly to the cyclopentano-perhydro-phenanthrene ring, decreases lipid raft order,and increases membrane polarity (Rentero et al., 2008; Schieffer, Naware, Bakun, &Bamezai, 2014) Interestingly, the nonsteroidal, anti-inflammatory drug aspirin has ahigh affinity for phospholipid membranes and partitions into the lipid head groups.This interaction impairs the molecular organization brought about by cholesteroland thus, leads to increased mobility in a lipid raft model (Alsop et al., 2015; Kyrikou,Hadjikakou, Kovala-Demertzi, Viras, & Mavromoustakos, 2004) The use of short-chain ceramides, i.e., C2-ceramide and C6-ceramide, decreases the plasma mem-brane lipid order and disrupts the lipid rafts as indicated by a reduction in the extent

of FRET between lipid raft markers (Gidwani, Brown, Holowka, & Baird, 2003)

Cholesterol is an integral component of lipid rafts in mammalian cell membranes,and membrane cholesterol levels are crucial in determining the stability and organi-zation of lipid rafts (Silvius, 2003) Thus, modifying the content of cholesterol in theplasma membrane is another option to disrupt the lipid raft and evaluate the function

of GPCRs The antifungal polyene antibiotics filipin (Brown & London, 2000;Drake & Braciale, 2001), nystatin (Oakley, Smith, & Engelhardt, 2009), and ampho-tericin (Wysoczynski et al., 2005) disrupt lipid rafts by binding and sequesteringcholesterol within the plasma membrane Pore-forming agents such as saponin(Hering, Lin, & Sheng, 2003; Schroeder, Ahmed, Zhu, London, & Brown, 1998),digitonin (Oliferenko et al., 1999), and streptolysin O (Fernandez-Lizarbe, Pascual,Gascon, Blanco, & Guerri, 2008) may also be used b-MCD is one of the mostfrequently used agents to deplete the endogenous cholesterol content of lipid rafts(Han et al., 2008; Yu et al., 2014, 2013) One advantage in using b-MCD is the avail-ability of a control for its use, i.e., a-MCD (Vial & Evans, 2005) Inhibitors of therate-limiting enzyme for cholesterol synthesis, the HMG-CoA reductase, may beused also to inhibit endogenous cholesterol biosynthesis These include drugssuch as simvastatin (Drake & Braciale, 2001) and lovastatin (Meszaros, Klappe,Hummel, Hoekstra, & Kok, 2011) A summary of protocols using these approaches

N-Way FRET microscopy can quantify interacting and noninteracting FRET pairs in

live cells (Hoppe, Scott, Welliver, Straight, & Swanson, 2013) The freely diffusible

FRET sensor Epac2-camps has been used to measure global cAMP responses of

lipid raft-associated receptors since it responds to changes in cAMP occurring

throughout the cytosolic compartment of cells (Agarwal et al., 2014) Moreover,

ver-sions of the Epac2-camps probe allow the selective targeting to lipid raft

(Epac2-MyrPalm) and nonraft (Epac2-CAAX) domains, which are useful in monitoring

local cAMP production near the plasma membrane (Agarwal et al., 2014) PALM,

as indicated above, and dSTORM have also been used to track the reorganization

Table 3 Common Strategies to Disrupt the Lipid Raft

Strategies Protocol

A Disruption of raft stability

Addition of gangliosides 10 e100 mM for 1 h ( Simons et al., 1999 )

Addition of PUFAs 50 mM, overnight ( Webb et al., 2000 )

Fumonisin B1 25 ug/mL, 48 e72 h ( Lipardi et al., 2000 )

7-cholesterol 35 e70 mM for 5 min to 2 h ( Schieffer et al.,

2014 ) Aspirin 10% For 30 e60 min in noncellular

experiments ( Alsop et al., 2015 )

C 2 - and C 6 -ceramide 32 and 8 mM, respectively ( Gidwani et al.,

2003 )

B Changing the cholesterol content

Filipin 2.5 e5 mg/mL for 15 min (stock solution:

5 mg/mL in ethanol) ( Drake & Braciale,

2001 ) Nystatin 20 e50 mg/mL for 1 h ( Oakley et al., 2009 )

Amphotericin 10 mg/mL for 1 h ( Wysoczynski et al., 2005 )

Saponin 5% in 20 mM phosphate buffer, pH 7.4, at

4
C for 10 min followed by extraction in 0.5% Triton X-100 at 4
C ( Hering et al.,

2003 ) Digitonin 0.003% for 30 min on ice ( Oliferenko et al.,

1999 ) Streptolysin O 500 ng/mL for 2 h ( Fernandez-Lizarbe

et al., 2008 ) b-MCD 2% for 1 h ( Han et al., 2008; Yu et al., 2014,

2013 )

10 mM for 1 h ( Simons, 1999 ) Lovastatin 1 mg/mL for 20 h ( Meszaros, 2011 )

Simvastatin Use 5 mg/mL for 12 h ( Drake & Braciale,

2001 )

PUFAs, Polyunsaturated Fatty Acids.

of lipid rafts (Tobin et al., 2014; Wu et al., 2013) Movement of single molecules inliving cells could also be tracked (single molecule tracking) (Scarselli et al., 2015).

In addition, fluorescent nanosensors that measure sodium in real time are reversibleand completely selective over other cations (Dubach, Das, Rosenzweig, & Clark,

2009) Real-time monitoring of sodium transport in response to stimulation or bition of GPCRs in intact or disrupted lipid rafts has become feasible

inhi-Current biochemical and biophysical techniques for studying GPCRs in lipidrafts, while helpful in many instances, are still rife with methodological drawbacksand limitations These include the requirement for cell membrane disruption, thereliance on antibodies that are specific for the GPCR of interest, the inability to studynative proteins, and the use of exogenous, often tagged, proteins Newer methodol-ogies that allow the study of GPCRs in their native form in intact cells are needed,such as the FRET biosensors for cAMP monitoring Meanwhile, the use of comple-mentary approaches that yield mutually supportive results may be the most judiciousway for drawing conclusions regarding the distribution and activity of GPCRs inlipid rafts

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Imaging GPCRs

trafficking and signaling

with total internal

reflection fluorescence

microscopy in cultured

neurons

2

Francheska Delgado-Peraza * ,x, Carlos Nogueras-Ortiz * ,

Agnes M Acevedo Canabal * ,x, Cristina Roman-Vendrell * ,{, Guillermo A Yudowski * ,x, 1

*Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,

San Juan, PR, USA

xDepartment of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico,

San Juan, PR, USA

{Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA

Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.10.002

© 2016 Elsevier Inc All rights reserved. 25

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellularevents occurring close and at the plasma membrane Over the last decade, we have seen asignificant increase in the number of publications applying TIRF microscopy to unravelsome of the fundamental biological questions regarding G protein-coupled receptors(GPCRs) function such as the mechanisms controlling receptor trafficking, quaternarystructure, and signaling among others Most of the published work has been performed inheterologous systems such as HEK293 and CHO cells, where the imaging surfaceavailable is higher and smoother when compared with the narrow processes or the smallercell bodies of neurons However, some publications have expanded our understanding ofthese events to primary cell cultures, mostly rat hippocampal and striatal neuronal cul-tures Results from these cells provide a bona fide model of the complex events con-trolling GPCR function in living cells We believe more work needs to be performed inprimary cultures and eventually in intact tissue to complement the knowledge obtainedfrom heterologous cell models Here, we described a step-by-step protocol to investigatethe surface trafficking and signaling from GPCRs in rat hippocampal and striatal primarycultures

The unique ability of total internal reflection fluorescence (TIRF) microscopy togenerate an evanescent field and excite fluorophores within a narrow optical section(100 nm) provides an ideal tool to investigate the multiple and dynamic eventsoccurring close to and at the plasma membrane of living cells with reducedphototoxicity and bleaching (Axelrod, 1981, 2003, 2008; Simon, 2009) Thischaracteristic resulted in the application of TIRF microscopy to multiple biologicalareas ranging from single molecule analysis to cell migration TIRF microscopy hasbeen particularly used to investigate protein translocation and trafficking to themembrane, vesicular events, and single molecule analysis among others(Mattheyses, Simon, & Rappoport, 2010; Reck-Peterson, Derr, & Stuurman,2010; Roman-Vendrell & Yudowski, 2015; Steyer & Almers, 2001) Since astro-cytes display a significant surface area, these cells have been used to visualizeand analyze various events such as the molecular mechanisms controlling vesicularrelease (Li, Agulhon, Schmidt, Oheim, & Ropert, 2013) However, the application ofTIRF microscopy to investigate G protein-coupled receptors (GPCRs) in neurons isstill very limited Our laboratory was among the first to use TIRF microscopy toinvestigate GPCRs in neurons (Yudowski, Puthenveedu, & von Zastrow, 2006).Our data showed that receptor recycling to the cell surface can be mediated bytwo different vesicular events, a rapid event without diffusion barriers and a slowevent with a kinetic similar to kiss and run exocytosis (Roman-Vendrell et al.,2014; Yudowski et al., 2006) These kiss and run events were not observed withthe Mu opioid receptor and were dependent on the carboxy terminal sequence ofthe B2 adrenergic receptor (Roman-Vendrell & Yudowski, 2015; Yu, Dhavan,Chevalier, Yudowski, & Zastrow, 2010) TIRF microscopy was also utilized todemonstrate that GPCR recycling after ligand-induced internalization is activelycontrolled by the activation of receptors still at the cell surface Interestingly,

identical results were observed in primary cell cultures and in heterologous systems

supporting the idea that heterologous systems can provide valuable information

(Bowman & Puthenveedu, 2015; Bowman et al., 2015; Roman-Vendrell, Yu, &

Yudowski, 2012; Yudowski, Puthenveedu, Henry, & Von Zastrow, 2009) Others

have utilized TIRF in neuronal cultures to investigate the pathways by which

recep-tors are recycled to the cell surface (Li et al., 2012) Our laboratory also utilized

TIRF microscopy to investigate endogenous GPCR signaling in hippocampal

neurons by loading neurons with calcium sensitive dyes and pharmacologically

acti-vating specific adrenergic receptors (Tzingounis, von Zastrow, & Yudowski, 2010)

More recently, we have investigated how cannabinoid receptor 1 interacts with

beta-arrestins at the cell surface of hippocampal neurons to regulate beta-arrestin

signaling (Flores-Otero et al., 2014) These are some of the applications of TIRF

mi-croscopy to investigate GPCRs The application of TIRF and superresolution

micro-scopy in combination with novel fluorescent tags and nanobodies should only

expand the toolbox available to further probe the biology of these highly relevant

receptors

To visualize GPCRs at the cell surface, receptors must be tagged with fluorophores

that are ideally resistant to quenching/photobleaching and with a significant quantum

yield (Shaner, Steinbach, & Tsien, 2005) One of the fluorescent tags widely utilized

in TIRF microscopy to investigate GPCRs is the pH-sensitive eGFP-variant super

ecliptic pHluorin (SEP) (Miesenbock, De Angelis, & Rothman, 1998) By attaching

the SEP molecule to the extracellular domain of GPCRs, receptors are highly visible

when they are located at the cell surface (neutral pH) and their fluorescence is

rapidly quenched in intracellular compartments such as endosomes (pH acidic)

The use of GPCRs tagged with SEP at the extracellular domain in TIRF microscopy

is an ideal approach to investigate events at the cell surface minimizing fluorescence

from receptors in intracellular compartments while reducing phototoxicity Other

available probes such as antibodies conjugated with quantum dots or SNAP tag

fusion proteins have been also utilized to investigate GPCRs surface trafficking

(Calebiro et al., 2013; Maurel et al., 2008; Mikasova, Groc, Choquet, & Manzoni,

2008; Reck-Peterson et al., 2010) However, their application to TIRF microscopy

is not as frequent as conventional genetically coded fluorophores

It is important to note that regardless of the florescent tag utilized, we strongly

recommend tagging receptors at their extracellular domain and compare their

function with wild-type receptors before any further analysis Only tagged receptors

with identical pharmacological and functional properties to wild-type receptors must

be used In our experience, tags in intracellular domains of GPCRs generally disrupt

their signaling and or trafficking, rendering nonphysiological behavior Finally, TIRF

1 Image acquisition 27

microscopy is not exempt from the general rules and pitfalls from live cell imaging,image acquisition, and analysis (Frigault, Lacoste, Swift, & Brown, 2009; Jaiswal &Simon, 2004; North, 2006; Schnell, Dijk, Sjollema, & Giepmans, 2012).

1.2.1 Cell culture

1 Striatal primary cultures obtained from embryonic day 17e18 Sprague-Dawleyrat embryos Alternatively, brain tissue can be purchased from BrainBits LLC(Springfield, IL)

2 Neuron culture media: Neurobasal medium supplemented with B27 (according

to manufacturer protocol) and 0.5 mM glutaMAXÔ (Life Technologies)

3 Imaging media such as Neurobasal nimus phenol red without serum and

supplemented with 20 mM HEPES (Life Technologies) (see Notes)

4 30 mm coverslips #1.5 thickness Coverslips must be acid washed and coated

with fresh poly-D-Lysine (Sigma) Glass bottom dishes (MatTek) can be alsoutilized They must be coated with PDL and the glass thickness must be 1.5

5 Transfection reagents, Lipofectamine 3000 (Life Technologies).

6 Hippocampal cultures were >90% pure as calculated by MAP2 and GFAPstaining as previously described (Yudowski et al., 2006, Yudowski, Olsen,Adesnik, Marek, & Bredt, 2013)

1.2.2 TIRF microscopy equipment and settings

1 Motorized Nikon (Melville, NY) Ti-E inverted microscope with a 100 chromat oil immersion TIRF objective lens (CFI Apo TIRF 100; Nikon), colorcorrection and a motorized stage with perfect focus (see Notes)

apo-2 Light source: 488 and 561 nm Coherent sapphire lasers (Coherent Inc Santa

Clara, CA) 50 and 100 mW lasers, respectively

3 Temperature control is utilized to keep cells at 37C with a stable Z stage and

objective warmer (Bioptechs, Butler, PA)

4 Interchangeable Coverslip Dish (Bioptechs) (http://www.bioptechs.com/Products/ICD/coverslipdish.html)

5 Camera: iXonEMþ DU897 back-illuminated electron multiplying chargedcoupled device sensor camera (Andor, Belfast, UK)

6 Readout speed: 10 Hz, exposure time: continuous 50e100 ms exposure forreceptor recycling, electron multiplying gain of 300, no binning, bit depth¼ 14bits, camera temperature set to minimum and laser power to 10% for 488 nm at

50 mW

1.3.1 Cell culture

1 Acid clean coverslips or glass bottom dishes by incubation in 1 M HCl shaking

overnight Rinse two times with abundant ddH O and once with 70% ethanol

Leave in 95% ethanol until ready to use Before coating, UV and dry in the

culture hood no less than 1 h

2 Coat acid clean coverslips or glass bottom dishes with freshly prepared

100mg/mL poly-D-lysine (PDL) for 2e4 h at 37C Wash 3 times with sterile

water and air dry in sterile environment It is important to prepare PDL fresh for

every use Old PDL solutions result in less than ideal cell attachment

3 Plate 250,000e300,000 neurons per 35 mm dish Neurons are transfected at 4e5

days in vitro (DIV) and imaged at 15 DIV or later Expression levels for receptor

trafficking is ideal between 14 and 25 DIV

4 Transfect cells with DNA constructs using Lipofectamine 3000 or Effectene

according to the manufacturer’s instructions We perform imaging after DIV 15

(High expression levels will impair observation of individual events They can

also result in trafficking artifacts such as reduced internalization) Lentivirus

can be also used to infect targeted cells In our experience, infected cells do not

look as healthy as transfected cells

5 Replace conditioned media with 2 mL of freshly prepared Opti-MEM with

HEPES 15e30 min before imaging sessions Important: Do not to allow

neurons to dry during this process A small amount of media must be left at the

dish covering all cells during the process

6 Incubate cells at 37C>10 min to allow acclimatization to the new media

7 Transfer neurons to the microscope.

1.3.2 Live cell imaging

1 At least 30 min before any acquisition, turn on the microscope and the

tem-perature controllers Turn on the laser key and let the laser warm up Important:

Objective and imaging chamber temperature must be controlled before and

during experiments Temperature must be 37C at the glass.

2 Select TIRF objective, add a drop of immersion oil (type LDF, RI:w1.515) and

carefully place cells on the stage (see Notes)

3 To reduce the effects of photobleaching, it is important to find and focus the cells

using transmission light first Then, find cells expressing tagged receptors using

epifluorescence and then switch to TIRF illumination (see Notes)

4 Add agonist diluted in warm imaging media by automated perfusion system or

manually outside the imaging area to minimize artifacts (see Notes)

5 Acquisition settings for endocytosis: 100e300 msec exposures every 2e3 s

Total time: 10e30 min

6 Acquisition settings for recycling: Continuous illumination and acquisition at

50e100 ms exposures for 1e2 min (see Notes)

7 Depolarization with 25 mM KCl is performed to test viability at the end of

ex-periments with Fluo-4

8 Imaging sessions will generate large amounts of data Careful data management

must be implemented in advance Standardized electronic notebooks or

spreadsheets are recommended

1 Image acquisition 29

1.4 NOTES

1 Healthy primary cultures are essential to obtain reliable and valid results.

Neurons must conserve their integrity, without blebbing or detachments

2 HEPES is used to maintain the pH constant for up to 45e60 min outside a CO2

incubator

3 High quality cDNA is highly desired for neuronal transfection Multiple

transfection agents are commercially available We utilize lipofectamine 2000

on DIV 4e5 and perform imaging on DIV > 15 This delay results in optimalexpression levels for TIRF imaging

4 Focal plane must be kept constant during imaging sessions.

5 It is very important that the cells grow in monolayer and are not more than

80e90% confluent on the day of imaging

6 It is very important that the bottom of the imaging dish is completely dry and

clean Any liquid or dirt will interfere during TIRF imaging

7 The most critical step is to find the exact angle for TIRF To align the laser

properly, focus on the plasma membrane You can find the cell sharp edges anduse them as reference

8 If ligands are added manually, extreme care is needed to prevent disturbing the

cells within the imaging area Controls should be performed to test the effects ofdimethyl sulfoxide and other solvents on surface fluorescence and basal cellactivity

9 Endocytosis should be visible within 1 or 2 min of agonist addition

Agonist-induced recycling can be observed 2e3 min after initial exposure A constantrate of vesicular fusion is generally observed atw10 min

Exocytotic events are easily identified by direct visualization of the abrupt increase(w1 s) in intensity at discrete points in the cell surface Image sequences can beanalyzed using the acquisition software available from the microscope or by usingthe public domain NIH Image program ImageJ/FIJI software, which is freely avail-able athttp://fiji.sc/Fiji Orthogonal views (kymographs) can be used to distinguishthese events from other vesicular event due to their characteristic kinetics and toquantify their frequency, decay kinetics, and location Maximum intensity fluores-cence can be extracted and plotted to demonstrate lateral diffusion and number ofexocytic molecules among others More recently, software specifically designed

to identify exocytic events has been developed such as the exocytosis detectionrecipe from SVCell (https://www.svcell.com/recipes/exocytosis-detection) Furtherdevelopment of this and other open source codes should provide better tools toextract and analyze exocytic events We recommend performing all the analysisblindly to reduce bias in the quantification

Endocytic events are intrinsically more challenging to analyze due to their lowersignal to noise ration Manual detection was initially utilized following specific