Tight junctions contain oligomeric protein assembly critical formaintaining blood–brain barrier integrity in vivo

Gwen McCaffrey,* William D. Staatz,* Carolyn A. Quigley,* Nicole Nametz,*Melissa J. Seelbach,* Chris R. Campos,*Tracy A. Brooks,� Richard D. Egleton� andThomas P. Davis*

*Department of Medical Pharmacology, University of Arizona College of Medicine, 1501 N. Campbell Ave, Tucson, Arizona, USA

�Bio5 Institute, Tucson, Arizona, USA

�Department of Pharmacology, Physiology and Toxicology, Marshall University School of Medicine, Huntington, West Virginia, USA

Abstract

Tight junctions (TJs) are major components of the blood–brain

barrier (BBB) that physically obstruct the interendothelial

space and restrict paracellular diffusion of blood-borne sub-

stances from the peripheral circulation to the CNS. TJs are

dynamic structures whose intricate arrangement of oligomeric

transmembrane and accessory proteins rapidly alters in re-

sponse to external stressors to produce changes in BBB

permeability. In this study, we investigate the constitutive

trafficking of the TJ transmembrane proteins occludin and

claudin-5 that are essential for forming the TJ seal between

microvascular endothelial cells that inhibits paracellular diffu-

sion. Using a novel, detergent-free OptiPrep density-gradient

method to fractionate rat cerebral microvessels, we identify a

plasma membrane lipid raft domain that contains oligomeric

occludin and claudin-5. Our data suggest that oligomerization

of occludin involves disulfide bond formation within trans-

membrane regions, and that assembly of the TJ oligomeric

protein complex is facilitated by an oligomeric caveolin scaf-

fold. This is the first time that distribution of oligomeric TJ

transmembrane proteins within plasma membrane lipid rafts

at the BBB has been examined in vivo. The findings reported

in this study are critical to understand the mechanism of

assembly of the TJ multiprotein complex that is essential for

maintaining BBB integrity.

Keywords: blood–brain barrier, lipid rafts, occludin, tight

junctions, trafficking.

J. Neurochem. (2007) 103, 2540–2555.

The blood–brain barrier (BBB) is the critical physical,metabolic, and immunological barrier that separates the CNSfrom the peripheral circulation (Reese and Karnovsky 1967;de Boer and Gaillard 2007). Anatomically, the BBB iscomposed of approximately 20 m2 of endothelial cells (per1.3 kg brain) that form the vascular microvessels of thebrain. Microvascular endothelial cells act in concert withadjacent astrocytes, pericytes, neurons, and the extracellularmatrix to create a ‘neurovascular unit’ to restrict the entry ofpotentially damaging blood-borne substances into the brain’sfragile microenvironment (Abbott et al. 2006). The BBB isemerging as an important therapeutic target because loss ofBBB integrity (i.e., leak) initiates and/or exacerbates numer-ous CNS and non-CNS diseases and pathologies includingAlzheimer’s disease (Kalaria 1996; Deane and Zlokovic2007), ischemia (Ilzecka 1996; Hom et al. 2007), multiplesclerosis (Trojano et al. 1992; Minagar et al. 2006), arthritis(Engblom et al. 2002), epilepsy (Oby and Janigro 2006),diabetes (Banks 2006; Hawkins et al. 2007) and peripheralinflammatory pain (Huber et al. 2001b, 2002; Brooks et al.2005, 2006). Recently, BBB permeability breaches havebeen shown to directly cause seizures both in humans and ina large animal model (Marchi et al. 2007). The ability torapidly seal breaches (or leaks) in the BBB, which occur

during pathological conditions, such as stroke, wouldcontribute significantly to the prevention of cerebral edemathat can lead to increased intracranial pressure and death(Petty and Lo 2002; Fraser 2006; Stamatovic et al. 2006b).

Cerebral microvascular endothelial cells that form theBBB are distinguished from most peripheral vascularendothelial cells by the absence of fenestrations, increasedmitochondria, reduced pinocytosis and the presence of tightjunctions (TJs) that connect apposing microvascular endo-thelial cell membranes (Hawkins and Davis 2005; de Boerand Gaillard 2007). Movement across the BBB can be eitherthrough (transcellular) or between microvascular endothelialcells (paracellular). Paracellular diffusion of water-soluble

Received May 31, 2007; revised manuscript received July 30, 2007;accepted August 16, 2007.Address correspondence and reprint requests to Gwen McCaffrey,

Ph.D., Department of Medical Pharmacology, College of Medicine,University of Arizona, P.O. Box 245050, 1501 N. Campbell Ave,Tucson, AZ 85724, USA. E-mail: gwenm@email.arizona.eduAbbreviations used: BBB, blood–brain barrier; EDT, 1,2-ethane-

dithiol; GFAP, glial fibrillary acidic protein; GLUT-1, glucose trans-porter; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TGFbRI,transforming growth factor-b receptor I; TJ, tight junction; ZO-1, zonulaoccludens-1.

Journal of Neurochemistry, 2007, 103, 2540–2555 doi:10.1111/j.1471-4159.2007.04943.x

2540 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

substances from the peripheral circulation to the brain andCNS is severely restricted by TJs. Inhibition of paracellulartransport of even small ions, such as Na+ and Cl) results in atransendothelial electrical resistance (TEER) of �2000 ohm-cm2, which is significantly greater than the 2–20 ohm-cm2

present within peripheral capillaries (Crone and Christensen1981).

Tight junctions are large, multiprotein complexes that notonly perform a ‘gate’ function in their selective inhibition ofparacellular transport between endothelial cells, but also a‘fence’ function in their inhibition of intermixing of luminal(blood side) and abluminal (brain side) plasma membraneproteins and lipids (Harhaj and Antonetti 2004; Feldmanet al. 2005). This maintenance of endothelial cell polarity byTJs is critical for effective BBB function, and it requires theasymmetric placement of specific luminal and abluminalnutrient and efflux transporters (Lippoldt et al. 2000; Tewesand Galla 2001; Hawkins et al. 2006). Oligomeric proteinassembly is believed to be an essential architectural feature ofTJs that renders them capable of performing these ‘gate’ and‘fence’ functions. Freeze-fracture replica electron micros-copy studies reveal TJs to be continuous, anastomosing,intramembranous particle strands or fibrils of �10 nmthickness that are thought to represent tightly packedoligomeric assemblies of integral membrane proteins (Shiv-ers et al. 1984; Fujimoto 1995; Mitic et al. 2000). It is thefusing of TJ strands between plasma membranes of adjacentendothelial cells that obliterates the interendothelial cleft andestablishes the physical barrier to paracellular diffusion(Sedlakova et al. 1999; Tsukita et al. 2001).

The transmembrane proteins critical for paracellularbarrier function at the BBB are occludin and claudin-5(Nitta et al. 2003; Harhaj and Antonetti 2004). Occludin andthe claudins share a common membrane topology, althoughthey have dissimilar primary sequences. Their N- and C-termini are cytoplasmic and they have four transmembranedomains and two extracellular loops that project into theparacellular space. Through their extracellular loops, occlu-din and claudin interact with homologous segments onapposing endothelial cell membranes to form the seal thatrestricts paracellular diffusion (Lacaz-Vieira et al. 1999; Wenet al. 2004). Occludin and claudin-5 are capable of self-association, and in vitro studies have shown that transmem-brane homodimers of these proteins are important features inthe assembly of TJs (Blasig et al. 2006). ZO-1 is the primarycytoplasmic protein associated with TJs. It links the C-termini of occludin and the claudins to the underlying actincytoskeleton (Furuse et al. 1994; Itoh et al. 1999).

Tight junction proteins are believed to be incorporatedwithin cholesterol-enriched regions of the plasma membrane(Kachar and Reese 1982; Stevenson and Goodenough 1984;Nusrat et al. 2000), and TJ function is modulated bycholesterol (Stankewich et al. 1996; Francis et al. 1999;Lambert et al. 2005). Physically distinct, cholesterol- and

sphingolipid-enriched regions of the plasma membrane,known as ‘lipid rafts,’ are increasingly being recognized asimportant sites of protein and lipid trafficking and signaltransduction (Simons and Toomre 2000; Helms and Zurzolo2004; Pike 2005). Interactions between cholesterol and thehydrocarbon chains of sphingolipids induce the lipids withinlipid rafts to become more tightly packed. This generates aliquid-ordered membrane domain that resists detergentsolubilization (Ahmed et al. 1997; Pike 2004) and promotesprotein oligomerization (Cunningham et al. 2003; Latif et al.2007).

Detailed biochemical understanding of key steps in TJprotein trafficking and oligomeric TJ protein assembly hasbeen hampered by the technical difficulty of isolating nativeTJ complexes. The differential distribution of TJ proteinsbetween detergent-soluble and detergent-insoluble fractionsis an established method to study TJ protein association withthe cytoskeleton (Nusrat et al. 2000; Stamatovic et al. 2003;Song et al. 2007). However, the use of detergent-containingmethods to isolate plasma membrane lipid raft domainsprecludes the ability to isolate native TJ complexes thatcontain oligomeric protein assemblies and the full comple-ment of associated accessory proteins to form the TJ (Pike2004; Ostrom and Insel 2006).

In this study, we employ the novel, neutral pH, detergent-free fractionation method of Macdonald and Pike (2005) toisolate plasma membrane lipid rafts from rat cerebralmicrovessels, and to examine the constitutive trafficking ofthe key TJ proteins occludin, claudin-5, and ZO-1 betweenplasma membrane domains and other subcellular compart-ments. Our data suggest that incorporation of preformed TJtransmembrane protein homodimers is an integral feature ofTJ complex assembly at the plasma membrane in vivo.

Materials and methods

Chemicals and reagents

OptiPrep was purchased from Accurate Chemical (Westbury, NY,

USA). EDTA-free Complete Proteinase Inhibitor was obtained from

Roche (Indianapolis, IN, USA). Criterion XT (10% Bis–Tris) gels,

20 · 3-morpholinopropanesulfonic acid running buffer, 4 · XT

sample loading buffer, 20 · XT reducing agent (tris(2-carboxyethyl)

phosphine hydrochloride, TCEP) and Precision Plus pre-stained

molecular weight markers were obtained from Bio-Rad (Hercules,

CA, USA). Polyvinylidene difluoride (0.45 lm) and Western

Lightning Chemiluminescence Reagent Plus were purchased from

Perkin-Elmer (Waltham, MA, USA). Blue autoradiography film was

bought from Genesee Scientific (San Diego, CA, USA). Bicinchon-

inic acid protein assay reagent and albumin standard were purchased

from Pierce (Rockford, IL, USA). The hydrophobic reducing agent

1,2-ethanedithiol (EDT), p-nitrophenyl phosphate and the b-N-acet-ylglucosaminidase assay kit were purchased from Sigma (St Louis,

MO, USA). All other chemicals and reagents were obtained from

either Sigma or Fisher Scientific (Fairlawn, NJ, USA).

Occludin oligomers at the BBB 2541

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

Antibodies

The goat polyclonal antibodies against occludin extracellular

microdomain (sc-27151), occludin N-terminus (sc-8145) and

Rab13 (sc-30374), and the rabbit polyclonal antibodies against

occludin internal region (sc-5562), flotillin-2 (sc-25507) and

nucleoporin (sc-25523) were obtained from Santa Cruz (Santa

Cruz, CA, USA). The mouse monoclonal antibodies against b-COP(G 6160), glial fibrillary acidic protein (GFAP) (G3893), sm-actin

(A2547) and b-actin (A-5316) were from Sigma. The mouse

monoclonal antibodies against occludin (33–1500), claudin-5 (35–

2500) and ZO-1 (33–9100), the rabbit polyclonal antibody against

occludin C-terminus (71–1500), and mouse primary antibody

isotype control antibody (08–6599) were purchased from Zymed

(South San Francisco, CA, USA). The mouse monoclonal antibody

against caveolin-1 (610406) was purchased from BD Transduction

Laboratories (San Jose, CA, USA). The rabbit polyclonal antibody

against GLUT-1 (400060) was from EMD Chemicals (San Diego,

CA, USA).

Animals

Female Sprague–Dawley rats (250–300 g) were purchased from

Harlan Sprague–Dawley (Indianapolis, IN, USA), housed under

standard 12 h light/12 h dark conditions, and given food and water

ad libitum. All treatment protocols were approved by the University

of Arizona Institutional Animal Care and Use Committee, and abide

by National Institutes of Health guidelines. Rat cerebral microvessel

isolations were optimized to use a n of 5 rats for each isolation. All

microvessel isolations for a particular dataset were performed at

least twice (n = 10 rats), and data presented are representative of

replicates performed.

Microvessel isolation

Animals were anesthetized with sodium pentobarbital (64 mg/kg)

and then subjected to transcardiac perfusion with 0.9% saline for

2 min. Following decapitation, brains were removed and immedi-

ately immersed in ice-cold Buffer A (15 mmol/L HEPES, pH 7.4,

containing 103 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2,

1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2 mmol/L phenylmeth-

ylsulfonyl fluoride (PMSF), 1 mmol/L Na3VO4, 1 mmol/L NaF and

1 mmol/L sodium pyrophosphate) with Roche EDTA-free Complete

Protease Inhibitor. The choroid plexi and meninges were removed,

and the cerebral hemispheres were homogenized in ice-cold Buffer

B (Buffer A supplemented with 25 mmol/L NaHCO3, 10 mmol/L

glucose, 1 mmol/L sodium pyruvate and 10 g/L 64K dextran). Five

milliliter of homogenate was mixed with 7 mL of 26% dextran,

centrifuged for 10 min at 5800 g (4�C) and the supernatant was

carefully removed by aspiration. The pellet was resuspended in

5 mL ice-cold Buffer B and subjected to a second dextran gradient

centrifugation. The resultant pellet was resuspended in 8 mL of ice-

cold Buffer B, and then filtered by gravity through a 70 lm mesh

filter (Becton Dickinson; Franklin, NJ, USA). Filtered homogenates

were pelleted by centrifugation for 10 min at 1500 g (4�C),resuspended in 3 mL ice-cold Buffer C (20 mmol/L Tris–HCl, pH

7.8, 250 mmol/L sucrose supplemented with 1 mmol/L CaCl2,

1 mmol/L MgCl2, Roche EDTA-free Complete Protease Inhibitor,

2 mmol/L PMSF, 1 mmol/L Na3VO4, 1 mmol/L NaF and 1 mmol/

L sodium pyrophosphate) and divided into aliquots for protein

assay, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS-PAGE) or density-gradient fractionation. Protein concentra-

tions were determined by bicinchoninic acid protein assay using

bovine serum albumin as the standard (Pierce; Rockford, IL, USA).

Confocal microscopy

Rat cerebral microvessels (isolated as described above) were spread

onto glass microscope slides and air-dried for 15 min at 25�C. Theywere then fixed with 3% formaldehyde, permeabilized with 0.1%

Triton X-100 and blocked in 1% bovine serum albumin and

immunostained, as previously described (Brooks et al. 2005).

Primary antibodies for occludin, claudin-5, and ZO-1 were used at

a dilution of 1 : 100, and Alexafluor 488-conjugated anti-mouse- or

anti-rabbit IgG (R&D Systems; Minneapolis, MN, USA) secondary

antibodies were used at 1 : 500. After washing in phosphate-

buffered saline, slides were sealed with Vectashield (Vector

laboratories Inc; Burlingame, CA, USA). Images were acquired

using an LSM 510 confocal laser-scanning microscope under an x63

oil-immersion objective and data was analyzed by LSM 5 software

(Zeiss; Thornwood, NY, USA). Microvessel morphology was

confirmed using phase contrast optics.

Microvessel fractionation

Rat cerebral microvessels (isolated as described above) were

fractionated by an adaptation of the detergent-free method of

MacDonald and Pike (2005). Briefly, isolated rat brain microvessels

that had been gently homogenized in Buffer C (20 mmol/L Tris–

HCl, pH 7.8, 250 mmol/L sucrose supplemented with 1 mmol/L

CaCl2, 1 mmol/L MgCl2, Roche EDTA-free Complete Protease

Inhibitor, 2 mmol/L PMSF, 1 mmol/L Na3VO4, 1 mmol/L NaF and

1 mmol/L sodium pyrophosphate) were passed 20 · through a

21 g · 1.5’ needle. Equal volumes of microvessel homogenate were

mixed with 50% OptiPrep in Buffer D (20 mmol/L Tris–HCl, pH

7.8, 250 mmol/L sucrose supplemented with Roche EDTA-free

Complete Protease Inhibitor, 2 mmol/L PMSF, 1 mmol/L Na3VO4,

1 mmol/L NaF and 1 mmol/L sodium pyrophosphate) and the

resultant 25% OptiPrep-microvessel homogenate was layered

beneath a discontinuous 0/5/10/15/20% OptiPrep gradient (prepared

in Buffer D) and centrifuged for 90 min at 52 000 g (4�C) in a SW-

28.1 rotor (Beckmann Coulter, Fullerton, CA, USA). One milliliter

fractions were collected from the top of the gradient, and fractions

were assayed for refractive index, protein content and enzyme

activity. Equal-volume aliquots of density-gradient fractions were

subjected to SDS-PAGE and western blot analysis.

Enzyme assays

Enzyme assays were carried out using 20–50 lL aliquots of each

density-gradient fraction and were performed in triplicate wells of

Greiner 96-well microtiter plates (Fisher Scientific). Assays were

started by adding 200 lL of enzyme assay mix to the gradient

fractions and incubating at 37�C for 3–16 h. Alkaline phosphatase

assay mix contained 0.2 mol/L sodium citrate, 2 mmol/L MgCl2,

2 mmol/L ZnCl2, pH 10.4, 0.1% Triton X-100 and 12 mmol/L

p-nitrophenyl phosphate. Alkaline phosphatase assays were stoppedby the addition of 75 lL of 1.5 mol/L acetic acid, and the

absorbance was read at 405 nm on a Labsystems Multiskan RC

plate reader (Fisher Scientific). b-N-acetylglucosaminidase activity

was measured using an assay kit from Sigma according to the

manufacturer’s instructions.

2542 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

SDS-PAGE and western blot analysis

Protein samples were mixed with XT sample loading buffer

(containing 1 · XT reducing agent TCEP) and heated for 10 min

at 70�C. In particular instances, protein samples were supplemented

with 10% SDS and 600 mmol/L EDT and heated for 10 min at

100�C. Protein samples and pre-stained molecular weight markers

were separated by SDS-PAGE on 10% Bis–Tris Criterion XT

precast gels using MOPS buffer, and electrophoretically transferred

to polyvinylidene difluoride membranes. Membranes were blocked

for 1 h at 25�C in 5% non-fat milk in 0.05% Tween-20- phosphate-

buffered saline buffer, and probed overnight at 4�C with primary

antibodies to occludin (1 : 1000), claudin-5 (1 : 500), ZO-1

(1 : 1000), caveolin-1 (1 : 500), nucleoporin (1 : 1000), cathepsin

D (1 : 1000), TGFbRI (1 : 1000), Rab13 (1 : 300), GLUT-1

(1 : 500), flotillin-2 (1 : 500), sm-actin (1 : 1000), b-actin(1 : 20,000) and GFAP (1 : 1000). After washing, the blots were

incubated with horseradish peroxidase-conjugated donkey anti-

rabbit, donkey anti-goat or sheep anti-mouse secondary antibody at

the appropriate dilutions. Protein bands were detected using Western

Lightning Chemiluminescence Reagent Plus and blue autoradio-

graphy film. Blot images were electronically scanned, and the

molecular weights of the protein bands estimated using Adobe

Photoshop and NIH Image J software.

Results

Characterization of isolated cerebral microvessels

Intact microvessels were isolated from rat brains using anestablished method (Huber et al. 2001b). The relativeenrichment of endothelial cell and TJ markers was deter-mined by examining the microvessel preparations by phasecontrast and immunofluorescence microscopy, and by ana-lyzing the microvessel homogenates on western blots(Fig. 1). Phase contrast showed that the isolates were highly

enriched for microvessels (Fig. 1a), free of intact neuronsand glial cells, and only occasionally associated withpericytes (arrowhead in Fig. 1a). Confocal microscopyrevealed staining for the TJ proteins occludin (Fig. 1b),claudin-5 (Fig. 1c) and ZO-1 (Fig. 1d) was restricted toisolated microvessels. There was no evidence of occludin inpericytes (arrowhead in Fig. 1b).

Western blots of whole brain homogenate and isolatedmicrovessel homogenate were probed for occludin, claudin-5, ZO-1, the endothelial cell marker glucose transporter(GLUT-1), the pericyte marker a-smooth-muscle actin (sm-actin) and GFAP (Fig. 1e). As compared to the loadingcontrol b-actin, microvessel homogenates were enriched foroccludin (fivefold), claudin-5 (sixfold) and ZO-1 (threefold).The endothelial cell marker GLUT-1 was enriched approx-imately ninefold. Microvessel homogenates were depleted ofsm-actin (13-fold) compared to whole brain. As reported byother laboratories (Pardridge et al. 1997; Yousif et al. 2007),we also found GFAP protein was present in our microvesselhomogenates. Collectively, these data indicate that ourmicrovessel isolates are enriched approximately five- toninefold for cerebral microvessels and contained minimalpericyte contamination. Because the pericytes and glial cellfragments did not express significant levels of TJ proteins(Fig. 1b–d), they should have little effect on the analysis ofTJ protein constitutive trafficking in endothelial cells at theBBB.

Isolation of plasma membrane lipid raft domains from

cerebral microvessels

To examine the constitutive trafficking of TJ proteinsbetween plasma membrane lipid rafts and intracellularcompartments in endothelial cells at the BBB, isolated

(a)

(b)

(c)

(d)

(e)

Fig. 1 Isolated microvessels are enriched for endothelial cells and

depleted of other cell types. Isolated cerebral microvessels were im-

aged by (a) phase contrast microscopy and by confocal microscopy

following immunostaining for (b) occludin, (c) claudin-5 and (d) ZO-1.

The same vessel is shown for contrast microscopy and immuno-

stained for occludin. Arrowheads indicate the position of an attached

pericyte. (e) Western blots of whole brain (Br) and isolated microvessel

(Mv) homogenates probed for occludin, claudin-5, ZO-1, and for the

glial cell protein GFAP, the pericyte marker a-smooth muscle actin,

and the endothelial cell marker GLUT-1. As a loading control, western

blots were stripped and reprobed for b-actin. Data shown are repre-

sentative of at least two independent experiments (n = 10–15 rats).

Occludin oligomers at the BBB 2543

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

cerebral microvessels were fractionated using the detergent-free method of MacDonald and Pike (2005). Microvesselswere homogenized in the presence of MgCl2 and CaCl2 (tostabilize lipid raft structures), and plasma membrane lipidrafts were separated from non-raft membranous componentsby flotation through a discontinuous 0–20% Optiprepgradient (prepared in the absence of MgCl2 and CaCl2).After centrifugation, 1 mL fractions were collected startingfrom the top of the gradient and assayed for refractive index,protein content, and enzyme activity. Equal-volume aliquotsof gradient fractions were subjected to SDS-PAGE andwestern blot analysis. To prevent over-estimation of proteinsassociated with low density, lipid-enriched membranedomains, the ‘constant volume’ (versus the ‘constantprotein’) mode of screening of density gradients was used(Macdonald and Pike 2005; Rudajev et al. 2005).

The density of each fraction was calculated from itsrefractive index, and values increased smoothly from1.038 g/mL (top of gradient) to 1.165 g/mL (bottom ofgradient) (Fig. 2a). The mean densities and standard errorsfor fractions obtained from three separate density gradients

prepared on different days show the consistency betweengradients. The protein profile (Fig. 2b) revealed there waslittle protein present in the low-density fractions, and that thebulk of the protein was found at the bottom of the gradient.Activity profiles for alkaline phosphatase (EC 3.1.3.1) and b-N-acetylglucosaminidase (EC 3.2.1.52), are shown in Fig. 2cand d. Alkaline phosphatase, a microvascular endothelial cellplasma membrane enzyme (Betz et al. 1980) associated withlipid rafts (Lupu et al. 1997), was found predominantly inthe low-density fraction 4 and to a lesser extent in fractions 7and 9 (Fig. 2c). Lipid rafts of different densities have beenshown to contain different levels of alkaline phosphatase inCaco-2 epithelial cells (Ellis et al. 1992), and our dataindicate that this may also occur in plasma membranes ofcerebral microvascular endothelial cells. In contrast, b-N-acetylglucosaminidase, a lysosomal enzyme, was found onlyat the bottom of the gradient. Western blots of gradientfractions 1–15 that were probed for TGFbRI and flotillin-2,which are known to be associated with plasma membranelipid rafts (Bickel et al. 1997; Zhang et al. 2005), revealedthese proteins to be found predominantly in fraction 7 and

(a)

(b)

(c)

(d)

(e)

Fig. 2 Characterization of detergent-free

OptiPrep density-gradients for the isolation

of plasma membrane lipid raft domains from

cerebral microvessels. Rat cerebral micro-

vessels homogenized in a neutral pH,

detergent-free buffer (containing MgCl2 and

CaCl2) were fractionated in a discontinuous

0–20% OptiPrep gradient (in the absence of

MgCl2 and CaCl2). One-mL fractions were

collected from the top of the gradient. (a)

Mean densities and standard errors from

three separate gradients on different days

(n = 15 rats). (b) Distribution of protein

across a representative gradient. (c) Distri-

bution of activity of the plasma membrane

lipid raft associated enzyme alkaline phos-

phatase across a representative gradient.

(d) Distribution of activity of the lysosomal

enzyme b-N-acetylglucosaminidase across

a representative gradient. (e) Western blots

of the first 15 fractions from a representa-

tive gradient probed for lipid raft markers

(TGFbRI, flotillin-2), the endosomal and

lysosomal protein cathepsin D, the nuclear

membrane protein nucleoporin and the

Golgi membrane protein b-COP. Bars at the

bottom of the figure indicate the distribution

of lipid raft and non-raft markers on the

gradient. Fraction 10 contains both raft and

non-raft markers. Data shown are repre-

sentative of at least two independent

experiments (n = 10–15 rats).

2544 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

fractions 7–8 (density 1.086–1.101 g/mL), respectively(Fig. 2e). Western blots of gradient fractions that wereprobed for cathepsin D detected the presence of thisendosomal and lysosomal enzyme predominantly in fraction10 and fractions 12–13 (Fig. 2e). The nuclear membraneprotein nucleoporin and the Golgi membrane protein b-COPwere also found primarily at the bottom of the gradient infractions 12–15 (density 1.134–1.161 g/mL). Thus, based ona combination of alkaline phosphatase activity and theantibody staining for flotillin-2 and TGFbRI, lipid rafts arepresent primarily in fractions 2–9. From the distribution of b-N-acetylglucosaminidase, nucleoporin, cathepsin D, and b-Cop, soluble proteins and internal cellular membranes appearto be present primarily in fractions 11–15 at the bottom of thegradient. Fraction 10 appears to contain a mixture of bothlipid raft material and material from internal cellularcompartments, based on the presence of lipid raft marker,flotillin, and non-raft marker, cathepsin D.

Collectively, these data show that the use of neutral pH,detergent-free homogenization, combined with OptiPrepdensity-gradient centrifugation, have allowed us to fraction-ate isolated cerebral microvessels to successfully separatedistinct plasma membrane lipid raft domains from intracel-lular membranous components. We therefore used thesemethods to examine the constitutive trafficking of TJ proteinsto plasma membrane lipid rafts at the BBB.

TJ protein isoforms are differentially distributed across

multiple plasma membrane and intracellular membrane

domains

Western blots of density-gradient fractions obtained byfractionating isolated rat cerebral microvessels were probedwith antibodies to occludin and claudin-5, and to theTJ-associated proteins ZO-1, caveolin-1 (Nusrat et al. 2000)and Rab13 (Marzesco et al. 2002; Morimoto et al. 2005). Toallow direct comparison of molecular weights of stainedbands between unfractionated and fractionated samples,aliquots of whole microvessel homogenates were subjectedto SDS-PAGE alongside the gradient fractions. Two impor-tant components of this analysis are that the microvesselhomogenates were prepared using a detergent-free neutral pHbuffer, and that aliquots of the entire homogenate, as opposedto detergent-soluble or detergent-insoluble material, weresubjected to SDS-PAGE. The pattern of protein bandsobserved in microvessel homogenates is, therefore, anindication of the relative expression of the TJ proteinisoforms within cerebral microvessels. In contrast, the patternof protein bands observed in gradient fractions is anindication of the relative distribution of these same TJ proteinisoforms within different plasma membrane domains andintracellular compartments. Antibody specificity was verifiedby control experiments in which the primary antibody wasomitted and only secondary antibody was used (data notshown). In addition, blots of microvessel homogenates and

density-gradient fractions (data not shown) that were probedwith a non-specific mouse IgG1 control antibody containedno visible bands. This non-specific antibody serves as anisotype control for the mouse IgG1 antibodies used to detectclaudin-5, ZO-1, and caveolin-1, as discussed below.

Rat claudin-5 has a predicted molecular weight of 23 kDa,and similar to claudin-4, claudin-5 forms detergent-stabledimers, trimers, tetramers, pentamers, and hexamers (Kojimaet al. 2002; Coyne et al. 2003; Mitic et al. 2003; Wang et al.2003). In porcine brain capillary endothelial cells multipleclaudin-5 isoforms have been detected, including a non-phosphorylated monomer (21 kDa), two phosphorylatedmonomers (25- and 31 kDa), and two phosphorylated dimers(42- and 62 kDa); whereas the monomers are detergent-soluble, the dimers resist detergent-extraction (Ishizaki et al.2003). Western blots of our whole microvessel homogenateand gradient fractions probed with a monoclonal antibody toclaudin-5 (Zymed 35–2500) detected prominent low-molec-ular weight bands (23- and 32 kDa) in non-raft fractions 12–15 (density 1.134–1.161 g/mL) at the bottom of the gradient.Additional higher molecular weight bands at 63- and155 kDa, which were not able to be detected in the wholemicrovessel homogenate, were concentrated by density-gradient fractionation and detected predominantly in fraction7 (density 1.086 g/mL) associated with plasma membranelipid rafts (Fig. 3a).

As expected, a monoclonal antibody to ZO-1 (Zymed 33–9100) detected a prominent band at approximately 223 kDain fractions 7–8 (density 1.086–1.101 g/mL), fraction 10(density 1.117 g/mL) and fractions 13–15 (density 1.134–1.153 g/mL) indicating that this accessory protein wasdistributed in both raft and non-raft membrane domains(Fig. 3b). The presence of weaker bands above the 223 kDaband suggests that multimeric forms of ZO-1 may also bepresent.

A subset of plasma membrane lipid rafts is associated withthe scaffolding protein caveolin-1 (Razani et al. 2002),which has been shown to bind occludin, be present within theinterendothelial space at TJs and to modulate occludin’spresence within TJs and BBB permeability (Nusrat et al.2000; Song et al. 2007). Western blots of microvesselhomogenates and gradient fractions were probed with amonoclonal antibody to caveolin-1 (BD Transduction610406) (Fig. 3c). Interestingly, low-molecular weight bandsat approximately 22 kDa were only found in fractions 13–15associated with ER/Golgi/nuclear membranes. However,high-molecular weight stained bands at approximately 72-and 89 kDa were found concentrated within fractions 5–10associated with plasma membrane lipid rafts. Caveolin-1 isreported to be phosphorylated (Minshall et al. 2003), and toform oligomers that are stable to heating at 70�C (Sargia-como et al. 1995), and these conditions were used ingenerating these western blots. The small GTPase, Rab13,is associated with intact TJ complexes in epithelial cells

Occludin oligomers at the BBB 2545

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

(Marzesco et al. 2002; Kohler et al. 2004) and with occludinendocytosis (Morimoto et al. 2005). In our gradient frac-tions, Rab13 staining was found predominantly in fractions 7and 8, with weaker staining also present in fraction 10(Fig. 3d).

Collectively, the presence of oligomeric claudin-5, alongwith significant levels of ZO-1 and Rab13, in fraction 7suggests that intact TJ complexes may be present in thisfraction. The data also suggest that assembly of theoligomeric TJ structure that is attached to the underlyingcytoskeleton through ZO-1 may be assisted by interactionwith an oligomeric caveolin scaffold.

Rat occludin has a predicted molecular weight of 59 kDa.Studies on occludin in mice, rats and humans have reported avariety of molecular weights for occludin that may be presentin cerebral microvessels because of alternative splicing(Mankertz et al. 2002) and post-translational modificationsincluding phosphorylation (Wong 1997; Feldman et al.2005; Kago et al. 2006; Stamatovic et al. 2006a; Seth et al.2007), ubiquitination (Traweger et al. 2002) and limitedproteolysis (Wachtel et al. 1999; Smales et al. 2003;

Lohmann et al. 2004; Zhu et al. 2006). Using a polyclonalantibody directed against the C-terminus (Zymed 71–1500),a set of bands that ranged in molecular weight fromapproximately 25- to 300 kDa was detected in both themicrovessel homogenate and the gradient fractions (Fig. 4a).The detection of these prominently stained bands suggeststhat significant amounts of multiple occludin isoforms maybe present in cerebral microvessels. The fact that none ofthese bands were found on blots probed with secondaryantibody in the absence of primary antibody (data not shown)suggests that these bands may be isoforms of occludin.According to the manufacturer, the polyclonal occludinantibody was raised against a peptide consisting of theC-terminal 150 amino acids of human occludin fused to GSTand then highly purified from rabbit antiserum by epitopeaffinity chromatography. Contained within the 150 aminoacid C-terminal occludin peptide is the conserved ‘occlu-din_ELL’ domain found in occludin and RNA polymerase IIelongation factors (Marchler-Bauer et al. 2005). A basiclocal alignment search tools search of all non-redundantGenBank CDS translations, PDB, SwissProt, PIR, and PRF

(a)

(b)

(c)

(d)

Fig. 3 Distribution of TJ protein isoforms

within distinct plasma membrane and

intracellular microdomains. Rat cerebral

microvessels were fractionated as de-

scribed for Fig. 2. Western blots of the first

15 fractions from a representative gradient

probed with antibodies to (a) claudin-5, (b)

ZO-1, (c) caveolin-1 and (d) Rab13. For

comparison, western blots of the unfrac-

tionated parental microvessel homogenate

are also shown (lane Mv). Densities, protein

concentrations and subcellular marker dis-

tributions are the same as in Fig. 2, and

bars at the bottom of the figure indicate the

distribution of lipid raft- and non-raft mark-

ers. *Indicates low-density (1.086 g/mL)

plasma membrane domain containing TJ

complexes. Data shown are representative

of at least two independent experiments

(n = 10–15 rats).

2546 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

databases for rat protein sequences that are homologous tothe C-terminal 150 amino acids of human occludin identifiedrat occludin as the only rat protein with significant sequencehomology (E = 3e)49) to the antigen. Short sequences fromfive other rat proteins, including the elongation factors ELL1-ELL3, were also identified as having weak sequencehomologies to the human occludin sequence (E < 1.0). Atmost, however, they contained 1–2 sequences of 3–4sequential amino acids identical to those in human occludin.While this does not exclude the possibility of the Zymed 71–1500 antibody weakly detecting proteins other than occludin,it is highly unlikely that this antibody would detect them asstrongly as it would occludin isoforms. While neither pre-immune serum nor the GST-occludin fusion protein iscommercially available for the Zymed 71–1500 rabbitpolyclonal antibody, a monoclonal antibody to occludin(Zymed 33–1500), prepared using the same GST-occludinfusion protein, is commercially available. On western blotsof microvessel homogenate (Fig. 4c) and density-gradientfractions (data not shown) probed with this monoclonalantibody (Zymed 33–1500), both monomeric and dimeric(Fig. 4c, lanes 1 and 2) forms of occludin were detected. Incontrast, parallel blots probed with a mouse IgG1 isotypecontrol mouse antibody (Zymed 08–6599) did not produceany stained bands (Fig. 4c, lane 3).

As shown in Fig. 3, data of the microvessel homogenates(lane Mv in Fig. 4a, c and d) are indicative of levels ofoccludin expression, whereas data from the gradient fractions(lanes 1–15 in Fig. 4a and e) are indicative of occludintrafficking. In the microvessel homogenates, the mostprominent bands observed were doublets at approximately53–65 and 35–37 kDa. Prominent single bands were alsovisible at approximately 110–120 and 25 kDa. In addition,three faint, high-molecular weight bands were detected atapproximately 140-, 260- and 300 kDa. Each of the occludinbands observed in the microvessel homogenate exhibited aunique pattern of distribution across the gradient, suggestingthat different occludin isoforms are targeted to specificplasma membrane and intracellular locations (Fig. 4a, d ande). High-molecular weight bands (approximately 260–300 kDa), that were weakly detected in the microvesselhomogenate, stained prominently in the gradient fractions 7–8, that are associated with plasma membrane lipid rafts. A110 kDa occludin band that was prominent in microvesselhomogenates appeared to be distributed primarily withinfractions 13–15, suggesting that this isoform is either free inthe soluble cytoplasm or is associated with membranes fromthe ER, Golgi, lysosomes, or nuclei. Weakly stainingoccludin bands at 110–120 kDa were also found in fractionsof density 1.086–1.101 g/mL and 1.117 g/mL, which areassociated with plasma membrane lipid rafts. A very densedoublet at approximately 53–65 kDa detected in the micro-vessel homogenate appeared to be resolved on the gradient toat least three distinct bands of approximate molecular

weights 53-, 59- and 65 kDa. Each of these bands exhibiteda unique distribution across the gradient. The 53- and 65 kDaoccludin isoforms were the most abundant, and theirpresence was concentrated in lipid raft associated fractions7–8 (density 1.086–1.101 g/mL). Interestingly, whereas asignificant amount of the 53 kDa occludin isoform appearedto be associated with light lipid rafts in fractions 5–6 atdensity 1.069–1.08 g/mL, neither the 59- nor the 65 kDaoccludin isoforms were targeted to these relatively lightplasma membrane domains. In contrast, appreciable amountsof the 59- and 65 kDa occludin isoforms were present at thebottom of the gradient in fractions 13–15. Only minoramounts of the 53 kDa occludin band were in fractions 11–13 and none was present at the bottom of the gradient.Strongly staining low-molecular weight bands, at approxi-mately 25-, 35- and 37 kDa, were also present in themicrovessel homogenate. Surprisingly, these low-molecularweight occludin bands were found almost exclusively withinthe lipid raft-containing gradient fractions 5–10. The low-molecular weight of these bands suggests that they may betruncated forms of occludin that retain enough of the C-terminus that they are able to be recognized by the polyclonalantibody (Zymed 71–1500), that was prepared against thelast 150 amino acids of the C-terminus of occludin (Fig. 4b).The absence of these low-molecular weight occludinisoforms at the bottom of the gradient, where lysosomalmembrane components are located, suggests that these areoccludin isoforms and are not products of lysosomaldegradation. Surprisingly, the 35 kDa occludin isoform wasthe most abundant protein species within the entire gradient.Similar to the 53 kDa occludin band, the 35 kDa occludinband was also detected in significant amounts in light lipidrafts (fractions 5–6). The 25 kDa occludin band was foundalmost exclusively in fractions 7–8 and 10. This distributionwas similar to that observed for the higher molecular weightoccludin band at 260–300 kDa.

Collectively, these data indicate that numerous occludinbands are selectively distributed between different lipid-enriched domains at the plasma membrane of microvascularendothelial cells at the BBB. The unique lipid-to-proteinratios of these different lipid-enriched domains, indicated bytheir distinct buoyant densities, ranging 1.069 to 1.117 g/mL,suggest a complexity of occludin isoform assembly withinlipid-enriched domains at the plasma membrane.

Occludin isoforms are detected by multiple antibodies

As described above, probing western blots with a rabbitpolyclonal antibody raised against a C-terminal peptide ofoccludin led to the identification of multiple bands that mayrepresent isoforms of occludin. To provide a more completecharacterization of these bands and to confirm that they areoccludin isoforms, western blots of microvessel homogenatesand OptiPrep gradient fractions were probed with antibodiesprepared against different amino acid sequences of this

Occludin oligomers at the BBB 2547

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

protein (Fig. 5). A goat polyclonal antibody (sc-5562)prepared against a peptide within an internal region ofoccludin (amino acids 136–411) detected a wide spectrum ofbands ranging from approximately 25–300 kDa (Fig. 5a).The most prominent band expressed within microvesselhomogenates was a wide band at approximately 53–65 kDa.

Within the gradient fractions, however, the most prominentbands detected were a high-molecular weight band atapproximately 300 kDa, and a low-molecular band atapproximately 25 kDa. The staining of both of the300 kDa and the 25 kDa occludin isoforms were pronouncedwithin fractions 7–10 (density 1.086–1.101 g/mL), previ-

(a)

(b)

(e)

(c) (d)

2548 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

ously shown to enriched for plasma membrane lipid rafts.Additional bands of lesser intensity at approximately 170,70–72 and 45 kDa were also found to be associated withplasma membrane lipid rafts.

A goat polyclonal antibody (sc-8145) prepared against theN-terminus of occludin also detected multiple bands rangingfrom approximately 30–300 kDa within microvessel homo-genates and their associated OptiPrep gradient fractions(Fig. 5b). A prominent doublet band at approximately 110–120 kDa was detected in the microvessel homogenate, and asimilar 110 kDa band was also present in the gradient

fractions 5–13, with most intense staining in fractions 7–8and 10. Additional higher molecular weight bands atapproximately 190- and 300 kDa, and lower molecularweight bands at approximately 70–72 kDa were alsodetected in both the microvessel homogenate and in theOptiPrep gradient fractions associated with plasma mem-brane lipid rafts. A band at approximate molecular weight65 kDa stained prominently in the microvessel homogenate.This 65 kDa occludin isoform was faintly detected within thegradient fractions 7–8, which are associated with plasmamembrane lipid rafts, and more prominently in fractions

(a)

(b)

Fig. 5 Occludin isoforms identified by different antibodies. Repre-

sentative western blots of OptiPrep gradient fractions of cerebral

microvessel homogenates were probed with anti-occludin antibodies

directed against (a) an internal region of occludin (sc-5562) and (b)

the N-terminus of occludin (sc-8145). Rat cerebral microvessels

were fractionated as described for Fig. 2. For comparison, western

blots of the unfractionated parental microvessel homogenate are

also shown (lane Mv). Small cartoons to the right of each blot depict

approximate locations of the antigenic peptides used to generate the

polyclonal anti-occludin antibodies. *Indicates low-density (1.086 g/

mL) lipid-enriched plasma membrane microdomain containing TJ

oligomeric assembly. Densities, protein concentrations and subcel-

lular marker distributions are the same as in Fig. 2. Bars at the

bottom of the figure indicate the distribution of lipid raft- and non-raft

markers. Data shown are representative of two independent exper-

iments (n = 10 rats).

Fig. 4 Distribution of occludin protein isoforms within distinct plasma

membrane and intracellular microdomains. Rat cerebral microvessels

were fractionated as described for Fig. 2. (a) Western blot of the first

fifteen fractions from a representative gradient probed with the Zymed

polyclonal antibody (71–1500) to the C-terminus of occludin. For

comparison, a western blot of the unfractionated parental microvessel

homogenate is also shown (lane Mv). (b) Cartoon depicting approxi-

mate location of antigenic peptide used to generate the polyclonal

anti-occludin antibody (Zymed 71–1500). (c) Western blots of unfrac-

tionated parental microvessel homogenate probed with the Zymed

monoclonal IgG1 antibody (33–1500) to the C-terminus of occludin

(lanes 1, 2) and mouse isotype control antibody (08–6599) (lane 3).

Western blot shown in lane 2 is a prolonged exposure of that shown in

lane 1; arrows indicate positions of monomeric, dimeric and oligomeric

isoforms of occludin. (d) Bar graph summarizing the relative expres-

sion of occludin isoforms in isolated rat cerebral microvessel homog-

enate shown in (a). (e) Plots show the relative distribution of occludin

isoforms between plasma membrane microdomains and other sub-

cellular compartments shown in (a). *Indicates low-density (1.086 g/

mL) plasma membrane microdomain containing TJ oligomeric

assembly. Densities, protein concentrations and subcellular marker

distributions are the same as in Fig. 2. Bars at the bottom of the figures

indicate the distribution of lipid raft- and non-raft markers. Data shown

are representative of three independent experiments (n = 15 rats).

Occludin oligomers at the BBB 2549

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

13–15 at the bottom of the gradient. Interestingly, theapparent inability of this N-terminal-occludin antibody todetect low-molecular bands within fractions associated withplasma membrane lipid rafts suggests that the low-molecularweight occludin isoforms detected by the other two antibod-ies have their antigenic N-terminus sites either removed orblocked. Collectively, the data from multiple occludinantibodies confirm that multiple occludin isoforms betweenapproximately 25–300 kDa are present within cerebralmicrovessels and that high-molecular weight dimeric andoligomeric forms of occludin are present within plasmamembrane lipid rafts of endothelial cells at the BBB.

TJ proteins form disulfide bonded oligomeric complexes

in TJ-enriched gradient fractions

The presence of occludin isoforms with apparent molecularweights between approximately 110–120 kDa and 300 kDasuggests the formation of dimeric and oligomeric TJ proteincomplexes, which are at least in part resistant to disulfidereduction by the hydrophilic reducing agent TCEP in thepresence of 2% SDS (Figs 4 and 5). Disulfide bondsembedded within hydrophobic cores of oligomeric proteinshave been identified and can be reduced using hydrophobicreducing agents, such as EDT (Zheng et al. 2006). Occludincontains cysteine residues within its hydrophobic transmem-brane domains that could be involved in disulfide linkages inthe native protein (Hua et al. 2003). Therefore, parallelaliquots of fraction 7 (density 1.086 g/mL) were prepared forSDS-PAGE under non-reducing conditions (2% SDS, heatedto 70�C for 10 min before electrophoresis) and understrongly reducing conditions (10% SDS, 600mmol/L EDT,heated to 100�C for 10 min before electrophoresis). Theresulting western blots were then probed with the C-terminusoccludin antibody (71–1500). As shown in Fig. 6, undernon-reducing conditions, a minimal amount of the mono-meric occludin isoform, of approximate molecular weight of53–65 kDa, was detected. A majority of the occludin wasfound in higher molecular weight (>100 kDa) oligomericisoforms. Under the strongly reducing conditions, however,there was a dramatic increase in the occludin monomers witha simultaneous decrease in higher molecular weight occludinoligomeric isoforms. Collectively, the data obtained with thehydrophilic reducing agent TCEP (Figs 4 and 5) and thehydrophobic reducing agent EDT (Fig. 6) confirm thatdetergent-free isosmotic OptiPrep fractionation of cerebralmicrovessels has allowed us to isolate TJ complexescontaining oligomeric protein assemblies of occludin thatare stabilized by disulfide linkages embedded within hydro-phobic regions of this transmembrane molecule.

Discussion

In this study, we report the use of a novel, neutral pH,detergent-free, isosmotic OptiPrep density-gradient method

(Macdonald and Pike 2005) to isolate plasma membrane lipidraft domains that contain native TJ complexes within thecontext of their normal plasma membrane environment. TJcomplexes isolated from intact cerebral microvessels con-tained high-molecular weight oligomeric assemblies ofoccludin and claudin-5. Co-localizing at the same densitywith these oligomeric transmembrane TJ proteins were theTJ-associated proteins ZO-1, caveolin-1, TGFbRI andRab13. Investigation of the constitutive trafficking of TJproteins revealed intracellular pools of apparent homodimersof occludin and claudin-5. Our data suggest that a key featureof TJ assembly at the BBB is the incorporation of preformedoccludin and claudin-5 homodimers that may be traffickedfrom intracellular locations to the plasma membrane inassociation with ZO-1 and caveolin-1.

Tight junctions at the BBB are remarkably dynamicstructures whose ability to restrict paracellular transport ofwater-soluble substances and ions rapidly changes inresponse to stressors, such as pain, inflammation and hypoxia(Huber et al. 2001a; Wolka et al. 2003; Hawkins and Davis2005). Previously, we used western blots and confocalmicroscopy to correlate global changes in TJ proteinexpression and localization within isolated cerebral micro-vessels with changes in paracellular permeability (Huberet al. 2001b, 2002; Hawkins et al. 2004, 2007; Brooks et al.2005, 2006). In this study, we used subcellular fractionationto analyze the constitutive trafficking of occludin, claudin-5

25015010075

50

37

20

250

Non-reduce

d

150

10075

50

37

20

kDa

Occludinoligomers

Occludinmonomers

kDa

Reduce

d

Fig. 6 Occludin forms disulfide bonded oligomers in TJs. Western

blots of parallel aliquots (7 lg per lane) of cerebral microvessel gra-

dient fraction 7 (density 1.086 g/mL) that were electrophoresed in the

absence of reducing agent and in the presence of the hydrophobic

reducing agent 1,2-ethanedithiol (EDT), and probed with the Zymed

polyclonal antibody (71–1500) directed against the C-terminus of oc-

cludin. Data shown are representative of two independent experiments

(n = 10 rats).

2550 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

and ZO-1 between plasma membrane domains and intracel-lular compartments in order to increase our understanding ofthe mechanism of assembly of TJ complexes within plasmamembrane lipid rafts at the BBB.

By using antibodies to different parts of occludin, we showthat multiple occludin isoforms (25–300 kDa) are distributedunder normal conditions amongst various plasma membranedomains and subcellular compartments within cerebralmicrovascular endothelial cells. Occludin isoforms areknown to be formed by mRNA splicing events and post-translational modifications including phosphorylation, ubiq-uitination and proteolytic cleavage (Harhaj and Antonetti2004; Feldman et al. 2005). Occludin splice variants formedby mRNA splicing in human HT-29 cells have predictedmolecular weights of 59 kDa (Type I), 53 kDa (Type II),32 kDa (Type III) and 57 kDa (Type IV) (Mankertz et al.2002). Phosphorylated forms of occludin (�72–75 kDa) areassociated with intact TJs (Wong 1997). Proteolytic cleavagesites have been identified that lead to the production ofoccludin peptides between 35 and 53 kDa (Wachtel et al.1999; Wan et al. 1999; Bojarski et al. 2004; Zeng et al.2004; Zhu et al. 2006).

Using an antibody directed against the C-terminus ofoccludin (Zymed 71–1500) to probe western blots ofgradient samples treated with the hydrophilic reducing agentTCEP, we detected monomeric (53–65 kDa), dimeric (100–120 kDa), and oligomeric (260–300 kDa) occludin isoforms.Whereas each of the monomeric occludin isoforms displayeda unique distribution in both high and low density membranedomains, dimeric occludin isoforms were found predomi-nantly in gradient fractions of high density. This suggests thata pool of occludin homodimers is present either in thecytosol or associated with ER/Golgi membranes. Interest-ingly, the C-terminus antibody showed that higher molecularweight oligomeric occludin isoforms were predominantly inlow-density membrane domains. The restriction of oligo-meric occludin isoforms to low-density, lipid-enrichedplasma membrane domains may reflect the importance ofthe plasma membrane lipid raft environment in both theassembly and maintenance of occludin-containing structuresof greater stability, such as the TJ. In addition to oligomericforms of occludin, low-molecular weight forms (25–37 kDa)were also found associated with plasma membrane lipid rafts.Their ability to be recognized by an antibody to the C-terminus, and not by an antibody to the N-terminus ofoccludin, reveals that constitutive intramembrane proteolysisat the TJ produces truncated, membrane-bound occludinisoforms that retain portions of the C-terminus.

Protein oligomers formed by disulfide links betweencysteine residues embedded within hydrophobic regions ofprotein molecules are known to be resistant to hydrophilicreducing agents, such as dithiothreitol and TCEP, that arecommonly used in SDS-PAGE (Zheng et al. 2006). Occludincontains such cysteine residues within its transmembrane

domains (Hua et al. 2003) that may be involved in formingTCEP-resistant homodimers. Treatment of our low-densitygradient fraction containing oligomeric occludin assemblies(fraction 7) with the hydrophobic reducing agent EDT for10 min at 100�C was sufficient to reduce all of the dimericand oligomeric forms of occludin to the 53–65 kDa mono-mer. In contrast, SDS-PAGE analysis in the absence ofreducing agent revealed that there was little free occludinmonomer (53–65 kDa) at the intact TJ complex, and themajority of the occludin was incorporated within 260–300 kDa oligomers, or to a lesser extent, within 110–120 kDa dimers.

Our investigation of the constitutive trafficking of claudin-5 within microvascular endothelial cells at the BBB revealedthat under normal conditions the majority of claudin-5appears to be stored, as low-molecular weight isoforms (23-and 32 kDa), either soluble in the cytoplasm or associatedwith ER/Golgi/nuclear membranes. Thus, the oligomericclaudin-5 we found associated with lipid rafts appears tocomprise only a small percentage of the total claudin-5 onour gradients. This difference in claudin-5 concentrations isreflected in the apparent absence of claudin-5 oligomers inour western blots of total microvessel homogenates. Pro-longed exposure of these blots, however, shows faintevidence of claudin-5 oligomers (data not shown). It is notknown whether this absence of detectability is because oflow levels of oligomeric claudin-5, or to the inability of theantibody to detect such oligomers. Ultrastructural andimmunofluorescent localization studies show claudin-5 tobe present not only within the junctional cleft but alsoassociated with the plasma membrane, with perijunctionalcytoplasmic plaques and distributed in a punctate mannerthroughout the cytoplasm (Dobrogowska and Vorbrodt 2004;Hawkins et al. 2004; Brooks et al. 2005; Cornford andHyman 2005). Immunoreplica electron microscopy showedthat claudin-5 was associated with TJ strands (Morita et al.1999). Consistent with this observation, the claudin-5 that wefound associated with plasma membrane lipid rafts wasoligomeric (63-, 155 kDa).

Increasingly it is being realized that proteins may ‘moon-light’ and perform multiple, unrelated functions. Determiningfactors for selection of a particular function may includecellular localization, oligomeric state and local concentrationof ligand, substrate, cofactor or product (Jeffery 2003). Thepatterns of occludin, claudin-5 and ZO-1 constitutivetrafficking that our density gradients provide may be givinginformation about multiple functions for each protein insteadof just one function. In addition, conclusions about occludintrafficking must be tempered with the realization thatdifferences in antibody affinity may be responsible for thedifferences in signal for occludin in lipid raft and non-raftfractions produced by different antibodies. Changes inprotein conformation, phosphorylation, limited proteolysisor other post-translational modifications may block the

Occludin oligomers at the BBB 2551

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

detection of occludin dimers by a specific antibody (e.g. N-terminal in lipid raft fractions or C-terminal in non-raftfractions 13–15). Despite differences in antibody specificity,an important point is that our method of fractionation allowsthe detection of occludin dimers in vivo. Moreover, based onantibody specificity, two distinct populations of occludindimers appear to be present in different locations (plasmamembrane lipid raft and internal non-raft membrane). Thesetwo populations of occludin dimers (lipid raft and non-raft)may serve different functions. For example, one populationof dimers may be involved in the assembly and/or disas-sembly of TJs, while the other population may havefunctions unrelated to the TJ.

Oligomerization of transmembrane TJ proteins is neces-sary to generate tightly packed TJ protein complexescapable of physically obliterating the interendothelial spaceand inhibiting paracellular transport between the blood andthe brain and thus forming the BBB. Moreover, rigid,oligomeric assemblies of transmembrane TJ proteinsattached to the underlying actin cytoskeleton by accessoryTJ proteins, such as ZO-1 would inhibit lateral diffusion ofproteins and lipids within the lipid bilayers and therebyprovide the ‘fence’ function by which TJs maintain cellpolarity (Shin et al. 2006). Occludin homodimer formationwas originally hypothesized following freeze-fracture elec-tron microscopic examination of TJ strands in endothelialcells. In these studies a C-terminus occludin antibodyproduced immunogold complexes on both the inner,protoplasmic (P-) and the outer, exoplasmic (E-) fracturefaces associated with TJ strands (Fujimoto 1995). Recentin vitro work using cotransfection of uniquely taggedoccludin and claudin-5 in eukaryotic cells demonstratedthat these proteins are capable of self-association and dimerformation (Blasig et al. 2006).

Biochemical confirmation of the oligomeric proteinassembly of TJs from the BBB has been elusive because ofdifficulty in purifying intact TJ complexes. TJs are incorpo-rated within lipid-enriched regions of the plasma membraneknown as ‘lipid rafts,’ which are recognized as cell signalingand membrane trafficking hotspots (Schneeberger and Lynch2004; Kohler and Zahraoui 2005; Pike 2005). The tightlypacked, ordered lipid structure of lipid rafts renders themresistant to detergent solubilization, while their high lipid-to-protein ratio causes them to have a very low-density.Consequently, lipid rafts are typically isolated using deter-gent extraction and sucrose density-gradient centrifugation.The standard definition of a lipid raft has, therefore, becomethe 1% Triton X-100-insoluble material that is present at theinterface of a 5%/30% sucrose (1.011–1.127 g/mL) stepgradient (Hope and Pike 1996; Pike 2004; Ostrom and Insel2006). However, detergent treatment of membranes cancause lipid-enriched domains to aggregate and form artifac-tual lipid raft structures not present in nature. Moreover, theintercalation of detergent molecules between proteins and

lipids within the lipid bilayer can remove proteins and lipids,which may influence the lipid-to-protein ratio, the tightnessof lipid packing and resident protein tertiary and quaternarystructure. Of particular concern is that the same detergentmay produce different concentrations of different membranefractions, and thus extraction of a specific protein may beinvestigator specific (Babiychuk and Draeger 2006). Deter-gent treatment with Triton X-100 also appears to selectivelyextract the outer leaflet of rafts, producing lipid raft structurescontaining an incomplete complement of inner leaflet lipids,which will influence the stability of transmembrane proteinoligomeric structures (Kurzchalia et al. 1995; Mayor andMaxfield 1995; Pike 2003, 2004; Schuck et al. 2003;Shogomori and Brown 2003). Detergent-free methods topurify lipid-enriched plasma membrane regions that usesonication and high pH may alter protein structure andenzyme activities of components within lipid rafts (Pike2004). To avoid difficulties with detergent-based methods, inthis study, we used the neutral pH, detergent-free density-gradient fractionation method of MacDonald and Pike(2005), which has recently been used to study partitioningof multimeric thyrotropin receptors within plasma membranelipid rafts (Latif et al. 2007).

In conclusion, our data show that TJ complexes, isolatedunder neutral pH, detergent-free conditions from intactcerebral microvessels, contain oligomeric assemblies ofoccludin and claudin-5. Furthermore, our data suggest thatthe maintenance of TJ complexes requires the constitutivetrafficking of occludin and claudin-5 homodimers to theplasma membrane, and the constitutive proteolysis ofoccludin to form membrane-attached occludin fragments.The interweaving of these low-molecular weight occludinisoforms amongst intact occludin and claudin-5 dimers maycreate a highly intricate barrier to paracellular transportacross the BBB. TJ disassembly may therefore involvehierarchical removal of occludin isoforms from the TJ,which would provide exquisite control of size selectivity ofdrugs, toxins and other molecules passing between endo-thelial cells to the brain and CNS. Controlling this passageis central to maintaining CNS homeostasis and health.These data are exciting because with the ability to isolateintact TJs, we can now investigate, at a molecular level, theeffects of pathological conditions on oligomeric assembly atthe TJ.

Acknowledgments

This work was supported by NIH grants R01-NS 39592, R01-

NS42652, R01-DA12684, and CA 09820-0251 to GM.

References

Abbott N. J., Ronnback L. and Hansson E. (2006) Astrocyte-endothelialinteractions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53.

2552 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

Ahmed S. N., Brown D. A. and London E. (1997) On the origin ofsphingolipid/cholesterol-rich detergent-insoluble cell membranes:physiological concentrations of cholesterol and sphingolipid in-duce formation of a detergent-insoluble, liquid-ordered lipid phasein model membranes. Biochemistry 36, 10944–10953.

Babiychuk E. B. and Draeger A. (2006) Biochemical characterization ofdetergent-resistant membranes: a systematic approach. Biochem.J. 397, 407–416.

Banks W. A. (2006) The dam breaks: disruption of the blood–brainbarrier in diabetes mellitus. Am. J. Physiol. Heart Circ. Physiol.291, H2595–H2596.

Betz A. L., Firth J. A. and Goldstein G. W. (1980) Polarity of the blood–brain barrier: distribution of enzymes between the luminal andantiluminal membranes of brain capillary endothelial cells. BrainRes. 192, 17–28.

Bickel P. E., Scherer P. E., Schnitzer J. E., Oh P., Lisanti M. P. andLodish H. F. (1997) Flotillin and epidermal surface antigen define anew family of caveolae-associated integral membrane proteins.J. Biol. Chem. 272, 13793–13802.

Blasig I. E., Winkler L., Lassowski B., Mueller S. L., Zuleger N., KrauseE., Krause G., Gast K., Kolbe M. and Piontek J. (2006) On the self-association potential of transmembrane tight junction proteins. CellMol. Life Sci. 63, 505–514.

de Boer A. G. and Gaillard P. J. (2007) Drug targeting to the brain. Annu.Rev. Pharmacol. Toxicol. 47, 323–355.

Bojarski C., Weiske J., Schoneberg T., Schroder W., Mankertz J.,Schulzke J. D., Florian P., Fromm M., Tauber R. and Huber O.(2004) The specific fates of tight junction proteins in apoptoticepithelial cells. J. Cell Sci. 117, 2097–2107.

Brooks T. A., Hawkins B. T., Huber J. D., Egleton R. D. and Davis T. P.(2005) Chronic inflammatory pain leads to increased blood–brainbarrier permeability and tight junction protein alterations. Am. J.Physiol. Heart Circ. Physiol. 289, H738–H743.

Brooks T. A., Ocheltree S. M., Seelbach M. J., Charles R. A., NametzN., Egleton R. D. and Davis T. P. (2006) Biphasic cytoarchitectureand functional changes in the BBB induced by chronic inflam-matory pain. Brain Res. 1120, 172–182.

Cornford E. M. and Hyman S. (2005) Localization of brain endothelialluminal and abluminal transporters with immunogold electronmicroscopy. NeuroRx 2, 27–43.

Coyne C. B., Gambling T. M., Boucher R. C., Carson J. L. and JohnsonL. G. (2003) Role of claudin interactions in airway tight junctionalpermeability. Am. J. Physiol. Lung Cell Mol. Physiol. 285,L1166–L1178.

Crone C. and Christensen O. (1981) Electrical resistance of a capillaryendothelium. J. Gen. Physiol. 77, 349–371.

Cunningham O., Andolfo A., Santovito M. L., Iuzzolino L., Blasi F. andSidenius N. (2003) Dimerization controls the lipid raft partitioningof uPAR/CD87 and regulates its biological functions. EMBO J. 22,5994–6003.

Deane R. and Zlokovic B. V. (2007) Role of the blood–brain barrier inthe pathogenesis of Alzheimer’s disease. Curr. Alzheimer Res. 4,191–197.

Dobrogowska D. H. and Vorbrodt A. W. (2004) Immunogold localiza-tion of tight junctional proteins in normal and osmotically-affectedrat blood–brain barrier. J Mol Histol 35, 529–539.

Ellis J. A., Jackman M. R. and Luzio J. P.(1992) The post-syntheticsorting of endogenous membrane proteins examined by thesimultaneous purification of apical and basolateral plasma mem-brane fractions from Caco-2 cells. Biochem. J. 283 (Pt 2), 553–560.

Engblom D., Ek M., Andersson I. M., Saha S., Dahlstrom M., JakobssonP. J., Ericsson-Dahlstrand A. and Blomqvist A. (2002) Induction ofmicrosomal prostaglandin E synthase in the rat brain endothelium

and parenchyma in adjuvant-induced arthritis. J. Comp. Neurol.452, 205–214.

Feldman G. J., Mullin J. M. and Ryan M. P. (2005) Occludin: structure,function and regulation. Adv. Drug Deliv. Rev. 57, 883–917.

Francis S. A., Kelly J. M., McCormack J., Rogers R. A., Lai J., Sch-neeberger E. E. and Lynch R. D. (1999) Rapid reduction of MDCKcell cholesterol by methyl-beta-cyclodextrin alters steady statetransepithelial electrical resistance. Eur. J. Cell Biol. 78, 473–484.

Fraser P. A. (2006) Can a broken barrier be repaired? J. Physiol. 573,287.

Fujimoto K.(1995) Freeze-fracture replica electron microscopy com-bined with SDS digestion for cytochemical labeling of integralmembrane proteins. Application to the immunogold labeling ofintercellular junctional complexes. J. Cell Sci. 108 (Pt 11), 3443–3449.

Furuse M., Itoh M., Hirase T., Nagafuchi A., Yonemura S., Tsukita S.and Tsukita S. (1994) Direct association of occludin with ZO-1 andits possible involvement in the localization of occludin at tightjunctions. J. Cell Biol. 127, 1617–1626.

Harhaj N. S. and Antonetti D. A. (2004) Regulation of tight junctionsand loss of barrier function in pathophysiology. Int. J. Biochem.Cell Biol. 36, 1206–1237.

Hawkins B. T. and Davis T. P. (2005) The blood–brain barrier/neuro-vascular unit in health and disease. Pharmacol. Rev. 57, 173–185.

Hawkins B. T., Abbruscato T. J., Egleton R. D., Brown R. C., HuberJ. D., Campos C. R. and Davis T. P. (2004) Nicotine increases invivo blood–brain barrier permeability and alters cerebral micro-vascular tight junction protein distribution. Brain Res. 1027, 48–58.

Hawkins R. A., O’Kane R. L., Simpson I. A. and Vina J. R. (2006)Structure of the blood–brain barrier and its role in the transport ofamino acids. J. Nutr. 136, 218S–226S.

Hawkins B. T., Lundeen T. F., Norwood K. M., Brooks H. L. andEgleton R. D. (2007) Increased blood–brain barrier permeabilityand altered tight junctions in experimental diabetes in the rat:contribution of hyperglycaemia and matrix metalloproteinases.Diabetologia 50, 202–211.

Helms J. B. and Zurzolo C. (2004) Lipids as targeting signals: lipid raftsand intracellular trafficking. Traffic 5, 247–254.

Hom S., Fleegal M. A., Egleton R. D., Campos C. R., Hawkins B. T. andDavis T. P. (2007) Comparative changes in the blood–brain barrierand cerebral infarction of SHR and WKY rats. Am. J. Physiol.Regul. Integr. Comp. Physiol. 292, R1881–R1892.

Hope H. R. and Pike L. J. (1996) Phosphoinositides and phosphoino-sitide-utilizing enzymes in detergent-insoluble lipid domains. Mol.Biol. Cell 7, 843–851.

Hua V. B., Chang A. B., Tchieu J. H., Kumar N. M., Nielsen P. A. andSaier Jr M. H. (2003) Sequence and phylogenetic analyses of 4TMS junctional proteins of animals: connexins, innexins, claudinsand occludins. J. Membr. Biol. 194, 59–76.

Huber J. D., Egleton R. D. and Davis T. P. (2001a) Molecular physiologyand pathophysiology of tight junctions in the blood–brain barrier.Trends Neurosci. 24, 719–725.

Huber J. D., Witt K. A., Hom S., Egleton R. D., Mark K. S. and DavisT. P. (2001b) Inflammatory pain alters blood–brain barrier per-meability and tight junctional protein expression. Am. J. Physiol.Heart Circ. Physiol. 280, H1241–H1248.

Huber J. D., Hau V. S., Borg L., Campos C. R., Egleton R. D. and DavisT. P. (2002) Blood–brain barrier tight junctions are altered during a72-h exposure to lambda-carrageenan-induced inflammatory pain.Am. J. Physiol. Heart Circ. Physiol. 283, H1531–H1537.

Ilzecka J. (1996) The structure and function of blood–brain barrier inischaemic brain stroke process. Ann. Univ. Mariae Curie Sklo-dowska [Med] 51, 123–127.

Occludin oligomers at the BBB 2553

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555

Ishizaki T., Chiba H., Kojima T., Fujibe M., Soma T., Miyajima H.,Nagasawa K., Wada I. and Sawada N. (2003) Cyclic AMP inducesphosphorylation of claudin-5 immunoprecipitates and expressionof claudin-5 gene in blood–brain-barrier endothelial cells via pro-tein kinase A-dependent and -independent pathways. Exp. CellRes. 290, 275–288.

Itoh M., Furuse M., Morita K., Kubota K., Saitou M. and Tsukita S.(1999) Direct binding of three tight junction-associated MAGUKs,ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J. CellBiol. 147, 1351–1363.

Jeffery C. J. (2003) Multifunctional proteins: examples of gene sharing.Ann. Med. 35, 28–35.

Kachar B. and Reese T. S. (1982) Evidence for the lipidic nature of tightjunction strands. Nature 296, 464–466.

Kago T., Takagi N., Date I., Takenaga Y., Takagi K. and Takeo S. (2006)Cerebral ischemia enhances tyrosine phosphorylation of occludinin brain capillaries. Biochem. Biophys. Res. Commun. 339,1197–1203.

Kalaria R. N. (1996) Cerebral vessels in ageing and Alzheimer’s disease.Pharmacol. Ther. 72, 193–214.

Kohler K. and Zahraoui A. (2005) Tight junction: a co-ordinator of cellsignalling and membrane trafficking. Biol. Cell 97, 659–665.

Kohler K., Louvard D. and Zahraoui A. (2004) Rab13 regulates PKAsignaling during tight junction assembly. J. Cell Biol. 165,175–180.

Kojima S., Rahner C., Peng S. and Rizzolo L. J. (2002) Claudin 5 istransiently expressed during the development of the retinal pigmentepithelium. J. Membr. Biol. 186, 81–88.

Kurzchalia T. V., Hartmann E. and Dupree P. (1995) Guilty by insolu-bility–does a protein’s detergent insolubility reflect a caveolarlocation? Trends Cell Biol. 5, 187–189.

Lacaz-Vieira F., Jaeger M. M., Farshori P. and Kachar B. (1999) Smallsynthetic peptides homologous to segments of the first externalloop of occludin impair tight junction resealing. J. Membr. Biol.168, 289–297.

Lambert D., O’Neill C. A. and Padfield P. J. (2005) Depletion of Caco-2cell cholesterol disrupts barrier function by altering the detergentsolubility and distribution of specific tight-junction proteins. Bio-chem. J. 387, 553–560.

Latif R., Ando T. and Davies T. F. (2007) Lipid rafts are triage centers formultimeric and monomeric thyrotropin receptor regulation. Endo-crinology 148, 3164–3175.

Lippoldt A., Kniesel U., Liebner S., Kalbacher H., Kirsch T., Wolburg H.and Haller H. (2000) Structural alterations of tight junctions areassociated with loss of polarity in stroke-prone spontaneouslyhypertensive rat blood–brain barrier endothelial cells. Brain Res.885, 251–261.

Lohmann C., Krischke M., Wegener J. and Galla H. J. (2004) Tyrosinephosphatase inhibition induces loss of blood–brain barrier integrityby matrix metalloproteinase-dependent and -independent path-ways. Brain Res. 995, 184–196.

Lupu C., Goodwin C. A., Westmuckett A. D., Emeis J. J., Scully M. F.,Kakkar V. V. and Lupu F. (1997) Tissue factor pathway inhibitor inendothelial cells colocalizes with glycolipid microdomains/caveo-lae. Regulatory mechanism(s) of the anticoagulant properties of theendothelium. Arterioscler. Thromb. Vasc. Biol. 17, 2964–2974.

Macdonald J. L. and Pike L. J. (2005) A simplified method for thepreparation of detergent-free lipid rafts. J. Lipid Res. 46,1061–1067.

Mankertz J., Waller J. S., Hillenbrand B., Tavalali S., Florian P.,Schoneberg T., Fromm M. and Schulzke J. D. (2002) Geneexpression of the tight junction protein occludin includes differ-ential splicing and alternative promoter usage. Biochem. Biophys.Res. Commun. 298, 657–666.

Marchi N., Angelov L., Masaryk T. et al. (2007) Seizure-promotingeffect of blood–brain barrier disruption. Epilepsia 48, 732–742.

Marchler-Bauer A., Anderson J. B., Cherukuri P. F. et al. (2005) CDD: aConserved Domain Database for protein classification. NucleicAcids Res. 33, D192–D196.

Marzesco A. M., Dunia I., Pandjaitan R., Recouvreur M., Dauzonne D.,Benedetti E. L., Louvard D. and Zahraoui A. (2002) The smallGTPase Rab13 regulates assembly of functional tight junctions inepithelial cells. Mol. Biol. Cell 13, 1819–1831.

Mayor S. and Maxfield F. R. (1995) Insolubility and redistribution ofGPI-anchored proteins at the cell surface after detergent treatment.Mol. Biol. Cell 6, 929–944.

Minagar A., Jy W., Jimenez J. J. and Alexander J. S. (2006) Multiplesclerosis as a vascular disease. Neurol. Res. 28, 230–235.

Minshall R. D., Sessa W. C., Stan R. V., Anderson R. G. and Malik A. B.(2003) Caveolin regulation of endothelial function. Am. J. Physiol.Lung Cell Mol. Physiol. 285, L1179–L1183.

Mitic L. L., Van Itallie C. M. and Anderson J. M. (2000) Molecularphysiology and pathophysiology of tight junctions I. Tightjunction structure and function: lessons from mutant animals andproteins. Am. J. Physiol. Gastrointest. Liver Physiol. 279,G250–G254.

Mitic L. L., Unger V. M. and Anderson J. M. (2003) Expression, solu-bilization, and biochemical characterization of the tight junctiontransmembrane protein claudin-4. Protein Sci. 12, 218–227.

Morimoto S., Nishimura N., Terai T., Manabe S., Yamamoto Y.,Shinahara W., Miyake H., Tashiro S., Shimada M. and Sasaki T.(2005) Rab13 mediates the continuous endocytic recycling of oc-cludin to the cell surface. J. Biol. Chem. 280, 2220–2228.

Morita K., Sasaki H., Furuse M. and Tsukita S. (1999) Endothelialclaudin: claudin-5/TMVCF constitutes tight junction strands inendothelial cells. J. Cell Biol. 147, 185–194.

Nitta T., Hata M., Gotoh S., Seo Y., Sasaki H., Hashimoto N., Furuse M.and Tsukita S. (2003) Size-selective loosening of the blood–brainbarrier in claudin-5-deficient mice. J. Cell Biol. 161, 653–660.

Nusrat A., Parkos C. A., Verkade P., Foley C. S., Liang T. W., Innis-Whitehouse W., Eastburn K. K. and Madara J. L.(2000) Tightjunctions are membrane microdomains. J. Cell Sci. 113 (Pt 10),1771–1781.

Oby E. and Janigro D. (2006) The blood–brain barrier and epilepsy.Epilepsia 47, 1761–1774.

Ostrom R. S. and Insel P. A. (2006) Methods for the study of signalingmolecules in membrane lipid rafts and caveolae. Methods Mol.Biol. 332, 181–191.

Pardridge W. M., Golden P. L., Kang Y. S. and Bickel U. (1997) Brainmicrovascular and astrocyte localization of P-glycoprotein.J. Neurochem. 68, 1278–1285.

Petty M. A. and Lo E. H. (2002) Junctional complexes of the blood–brain barrier: permeability changes in neuroinflammation. Prog.Neurobiol. 68, 311–323.

Pike L. J. (2003) Lipid rafts: bringing order to chaos. J. Lipid Res. 44,655–667.

Pike L. J. (2004) Lipid rafts: heterogeneity on the high seas. Biochem. J.378, 281–292.

Pike L. J. (2005) Growth factor receptors, lipid rafts and caveolae: anevolving story. Biochim. Biophys. Acta 1746, 260–273.

Razani B., Woodman S. E. and Lisanti M. P. (2002) Caveolae: from cellbiology to animal physiology. Pharmacol. Rev. 54, 431–467.

Reese T. S. and Karnovsky M. J. (1967) Fine structural localization of ablood–brain barrier to exogenous peroxidase. J. Cell Biol. 34,207–217.

Rudajev V., Novotny J., Hejnova L., Milligan G. and Svoboda P. (2005)Dominant portion of thyrotropin-releasing hormone receptor isexcluded from lipid domains. Detergent-resistant and detergent-

2554 G. McCaffrey et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555� 2007 The Authors

sensitive pools of TRH receptor and Gqalpha/G11alpha protein.J. Biochem. (Tokyo) 138, 111–125.

Sargiacomo M., Scherer P. E., Tang Z., Kubler E., Song K. S., SandersM. C. and Lisanti M. P. (1995) Oligomeric structure of caveolin:implications for caveolae membrane organization. Proc. Natl Acad.Sci. USA 92, 9407–9411.

Schneeberger E. E. and Lynch R. D. (2004) The tight junction: a mul-tifunctional complex. Am. J. Physiol. Cell Physiol. 286,C1213–C1228.

Schuck S., Honsho M., Ekroos K., Shevchenko A. and Simons K. (2003)Resistance of cell membranes to different detergents. Proc. NatlAcad. Sci. USA 100, 5795–5800.

Sedlakova R., Shivers R. R. and Del Maestro R. F. (1999) Ultrastructureof the blood–brain barrier in the rabbit. J. Submicrosc. Cytol.Pathol. 31, 149–161.

Seth A., Sheth P., Elias B. C. and Rao R. (2007) Protein Phosphatases2A and 1 Interact with Occludin and Negatively Regulate theAssembly of Tight Junctions in the CACO-2 Cell Monolayer.J. Biol. Chem. 282, 11487–11498.

Shin K., Fogg V. C. and Margolis B. (2006) Tight junctions and cellpolarity. Annu Rev Cell Dev Biol 22, 207–235.

Shivers R. R., Betz A. L. and Goldstein G. W. (1984) Isolated rat braincapillaries possess intact, structurally complex, interendothelialtight junctions; freeze-fracture verification of tight junction integ-rity. Brain Res. 324, 313–322.

Shogomori H. and Brown D. A. (2003) Use of detergents to studymembrane rafts: the good, the bad, and the ugly. Biol Chem 384,1259–1263.

Simons K. and Toomre D. (2000) Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 1, 31–39.

Smales C., Ellis M., Baumber R., Hussain N., Desmond H. and StaddonJ. M. (2003) Occludin phosphorylation: identification of an oc-cludin kinase in brain and cell extracts as CK2. FEBS Lett. 545,161–166.

Song L., Ge S. and Pachter J. S. (2007) Caveolin-1 regulates expressionof junction-associated proteins in brain microvascular endothelialcells. Blood 109, 1515–1523.

Stamatovic S. M., Keep R. F., Kunkel S. L. and Andjelkovic A. V.(2003) Potential role of MCP-1 in endothelial cell tight junction‘opening’: signaling via Rho and Rho kinase. J. Cell Sci. 116,4615–4628.

Stamatovic S. M., Dimitrijevic O. B., Keep R. F. and Andjelkovic A. V.(2006a) Protein kinase Calpha-RhoA cross-talk in CCL2-inducedalterations in brain endothelial permeability. J. Biol. Chem. 281,8379–8388.

Stamatovic S. M., Dimitrijevic O. B., Keep R. F. and Andjelkovic A. V.(2006b) Inflammation and brain edema: new insights into the roleof chemokines and their receptors. Acta Neurochir. Suppl. 96,444–450.

Stankewich M. C., Francis S. A., Vu Q. U., Schneeberger E. E. andLynch R. D. (1996) Alterations in cell cholesterol content modulateCa(2 + )-induced tight junction assembly by MDCK cells. Lipids31, 817–828.

Stevenson B. R. and Goodenough D. A. (1984) Zonulae occludentes injunctional complex-enriched fractions from mouse liver: pre-liminary morphological and biochemical characterization. J. CellBiol. 98, 1209–1221.

Tewes B. J. and Galla H. J. (2001) Lipid polarity in brain capillaryendothelial cells. Endothelium 8, 207–220.

Traweger A., Fang D., Liu Y. C., Stelzhammer W., Krizbai I. A., FresserF., Bauer H. C. and Bauer H. (2002) The tight junction-specificprotein occludin is a functional target of the E3 ubiquitin-proteinligase itch. J. Biol. Chem. 277, 10201–10208.

Trojano M., Manzari C. and Livrea P. (1992) Blood–brain barrierchanges in multiple sclerosis. Ital. J. Neurol. Sci. 13, 55–64.

Tsukita S., Furuse M. and Itoh M. (2001) Multifunctional strands in tightjunctions. Nat. Rev. Mol. Cell Biol. 2, 285–293.

Wachtel M., Frei K., Ehler E., Fontana A., Winterhalter K. and Gloor S.M.(1999) Occludin proteolysis and increased permeability inendothelial cells through tyrosine phosphatase inhibition. J. CellSci. 112 (Pt 23), 4347–4356.

Wan H., Winton H. L., Soeller C. et al. (1999) Der p 1 facilitatestransepithelial allergen delivery by disruption of tight junctions.J. Clin. Invest. 104, 123–133.

Wang F., Daugherty B., Keise L. L., Wei Z., Foley J. P., Savani R. C. andKoval M. (2003) Heterogeneity of claudin expression by alveolarepithelial cells. Am. J. Respir. Cell Mol. Biol. 29, 62–70.

Wen H., Watry D. D., Marcondes M. C. and Fox H. S. (2004) Selectivedecrease in paracellular conductance of tight junctions: role of thefirst extracellular domain of claudin-5. Mol. Cell. Biol. 24,8408–8417.

Wolka A. M., Huber J. D. and Davis T. P. (2003) Pain and the blood–brain barrier: obstacles to drug delivery. Adv. Drug Deliv. Rev. 55,987–1006.

Wong V. (1997) Phosphorylation of occludin correlates with occludinlocalization and function at the tight junction. Am. J. Physiol. 273,C1859–C1867.

Yousif S., Marie-Claire C., Roux F., Scherrmann J. M. and Decleves X.(2007) Expression of drug transporters at the blood–brain barrierusing an optimized isolated rat brain microvessel strategy. BrainRes. 1134, 1–11.

Zeng R., Li X. and Gorodeski G. I. (2004) Estrogen abrogates trans-cervical tight junctional resistance by acceleration of occludinmodulation. J. Clin. Endocrinol. Metab. 89, 5145–5155.

Zhang X. L., Topley N., Ito T. and Phillips A. (2005) Interleukin-6regulation of transforming growth factor (TGF)-beta receptorcompartmentalization and turnover enhances TGF-beta1 signaling.J. Biol. Chem. 280, 12239–12245.

Zheng J., Du G. G., Anderson C. T., Keller J. P., Orem A., Dallos P. andCheatham M. (2006) Analysis of the oligomeric structure of themotor protein prestin. J. Biol. Chem. 281, 19916–19924.

Zhu L., Li X., Zeng R. and Gorodeski G. I. (2006) Changes in tightjunctional resistance of the cervical epithelium are associatedwith modulation of content and phosphorylation of occludin 65-kilodalton and 50-kilodalton forms. Endocrinology 147, 977–989.

Occludin oligomers at the BBB 2555

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2540–2555