The fodrin-ankyrin cytoskeleton of choroid
plexus preferentially colocalizes with apical
Na+K(+)-ATPase rather than with basolateral
anion exchanger AE2.
S L Alper, … , D Brown, D Drenckhahn
J Clin Invest. 1994;93(4):1430-1438. https://doi.org/10.1172/JCI117120.
A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or
basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.
Research Article
Find the latest version:
The Fodrin-Ankyrin
Cytoskeleton
of Choroid Plexus
Preferentially Colocalizes
with Apical
Na+K+-ATPase
Rather than with Basolateral
Anion Exchanger AE2
Seth L. Alper,*11AlanStuart-Tilley,*Charles F.Simmons, Dennis
Brown,Ol
andDetlevDrenckhahn***MolecularMedicine and RenalUnits, Beth IsraelHospital;tDivision ofNeonatology, TheChildren'sHospital; RenalUnit, Massachusetts General Hospital;
IlDepartments
ofCellBiologyand'Pathology, HarvardMedical School, Boston,Massachusetts02215;and**The AnatomicalInstitute, University of Wilrzburg, 97070 W~rzburg, Germany
Abstract
Aunique feature ofthe choroid plexus as asingle-layer epithe-lium is its localization of Na+K+-ATPase at its apical (lu-menal) surface. In contrast,a band 3 (AE1)-relatedanion ex-changer proteinhasbeen localizedto thebasolateralsurface of the choroid plexus. Both Na+K+-ATPase and AEI in other tissues have beenshown to bind via ankyrin to the spectrin-ac-tin-based membrane cytoskeleton. Since linkage of integral membrane proteinsto the membranecytoskeletonisimportant for their restrictiontospecialized domainsof the cell surface, weinvestigated the polarity ofthe choroid plexusmembrane cytoskeleton. We developed isoform-specificantibodies to con-firm the identity of choroidplexus band3-related polypeptide asAE2. Wedemonstrated that ankyrin, fodrin/spectrin, actin, myosin, and a-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K+-ATP-aserather thanwithbasolateral anion exchanger AE2. Colchi-cineadministrationdidnotalter the polarity ofapical cytoskele-talandtransportproteinsorbasolateral AE2inchoroidplexus,
suggestingthatbiosynthetictargeting oftheseproteins is not microtubule dependent. In choroid plexus papilloma, Na+K+-ATPase and AE2weredecreased inamount and failed topreserve their polarizeddistributions.(J. Clin. Invest.1994.
93:1430-1438.)Key words: choroid plexus-epithelial polarity
*anionexchanger 2 .sodium-potassiumadenosine
triphospha-tase *cytoskeleton
Introduction
Thegreatmajority of plasma membrane proteins examinedto date inpolarized epithelia display polarity ofexpression. The
mechanismsofplasma membraneprotein targetingremain un-known.Initialexperimentsfocusedonthe searchfortargeting
signals within theprimary sequences ofthepolarized mem-braneproteins.Thoughoperationally defined targeting signals
have indeed been found in a small number ofsingle-span membrane proteins, nonehas beendefinedtodatefor
multi-ple-span membrane transport proteins. Whereas consensus targetingsequenceshavebeenidentified for organellar
mem-Portions ofthis work haveappearedin abstract form(1991.KidneyInt. 38:71la).
AddresscorrespondencetoDr.Seth L.Alper,Molecular Medicine
Unit,Beth IsraelHospital,330BrooklineAvenue, Boston,MA 02215.
Receivedfor publication 12August 1993and inrevisedform25
October1993.
braneproteins,such consensus sequenceshave not been found
forplasmalemmal proteins(1).
In addition to targeting signals within protein sequences, othertargeting mechanismshavebeen investigated.These in-cludeheterotrimeric(2) and low molecular weight G proteins (3)asvesicle targetinganddockingagentsand selective
reten-tionand stabilization byassociation via ankyrinto the
spec-trin/actin-based membrane cytoskeleton (4, 5). The latter
mechanism,inparticular,has been invokedtoexplainthe po-larized steadystatedistribution ofanion exchanger1 (AE 1)' in the basolateral membrane of renalcollectingduct intercalated cells(5) and oftheNa+K+-ATPase in thebasolateral mem-braneofMadin-Darbycanine kidney (MDCK) cells (4) and kidney tubules (6, 7). One clonal lineofMDCK cells appears to deliver nascent Na+K+-ATPase randomly to basolateral andapicalcellsurfacesbutselectively stabilizestheproteinat thebasolateral surface, apparentlythroughbindingtoankyrin (4,8).
In mostpolarized epithelialcells,immunoreactive ankyrin
isconcentratedatthe basolateral membrane (5, 9). However, several celltypes expressankyrin-associatedP-type ATPases in theapicalmembrane. Forexample, theapicovesicular localiza-tionoftheH+K+-ATPaseinrabbitgastric gland parietalcells appears to beaccompaniedbyapical localization ofimmunore-activeankyrin
(10).
Another celltypewhichexpressesapical ankyrin is the retinal pigment epithelium, in whichNa+ K+-ATPase colocalizestotheapical surface(11). How-ever, thisdouble-layer epithelium is uniquein that itsapical surface isnotlumenal but ratherisinN-CAM-stabilized con-tactwith photoreceptorcells( 12).Thechoroid plexus,in
con-trast,isasimple, single-layer
epithelium
withalumenalapicalsurface. Itis theonly suchepitheliumwhichexpressesapical
Na+K+-ATPase.Interestingly,thechoroidplexusalso secretes
apicallyinto the
cerebrospinal
fluid (CSF)transthyretin,
ceru-loplasmin,andcystatin C,allproteinswhicharesecreted baso-laterally by hepatocytes (13-15). ThecDNAs which encode these secretedproteinshave allbeencloned from bothchoroid
plexus andfrom liverand have beenfoundnot todifferin the twocelltypes(16).Inaddition, peptidemap,
immunochemi-cal( 17, 18),andtranscript analyses(19)haveindicatedthat choroidplexusexpressesthea1 subunit of the
Na+K+-ATP-ase, aswellas,BI and
32
subunits.Theunusualapical
expres-sion andsecretionofthesepolypeptides, biochemically
indistin-guishablefrom their basolateralcounterpartsinother
epithelial
tissues, hasledto the
hypothesis
thatthe choroidplexushas undergonea"reversal" ofelementsofitsprotein-sorting
mech-anism.In addition to the Na+K+-ATPase, another membrane transportprotein expressedin abundance inchoroid
plexus
is 1. Abbreviations usedin thispaper: AEl, anion exchanger 1;CSF, cerebrospinal fluid;MDCKcells, Madin-Darbycaninekidneycells.1430 Alper, Stuart-Tilley,Simmons, Brown,and Drenckhahn J. Clin.Invest.
©TheAmerican SocietyforClinicalInvestigation,Inc.
0021-9738/94/04/1430/9 $2.00
the band 3-relatedchloride/bicarbonate exchanger AE2 (20, 21). AE2 shares with the erythroid anion exchanger (AE1, band 3) conserved sequences within their cytoplasmic do-mains which havebeen proposed asbindingsites for nonery-throid homologs oferythroidcytoskeletal proteins,including ankyrin (22, 23). The choroid plexuspresents asituation in which aknownankyrin-binding protein,Na+K+-ATPase, is localized apically (24, 25) whilea proposed ankyrin-binding protein, AE2, is localized basolaterally (21 ).
Inthis paperwecharacterize isoform-specific antibodiesto AE2andusethemtoconfirm AE2asthe band 3-related pro-tein of the basolateral membrane of the choroid plexus. We localize AE2 and the Na+K+-ATPasetoopposite surfaces of thechoroid plexusepithelium. We show thatimmunoreactive forms ofankyrin, spectrin, and actin allareconcentrated at the apical membrane, away from AE2, and together with Na+K+-ATPase. Inspite of this"reversed polarity" of the an-kyrin/spectrin cytoskeleton, the microtubule organizing center appears tobeconventionally situated intheapicalpole of the cell(26), asjudged by thepreservation onlyof apical microtubules after in vivo colchicine administration. Interest-ingly, colchicine hadnoeffecton the steady statedistribution ofanyof theapical proteins of the choroid plexus studied, in contrast tothe significant redistribution of apical hydrolases and cytoskeletal proteins produced in the brush border of ileum (26) and the redistribution of apicalgp330 in the renal proximal tubule (27). Lastly, we show that both apical Na+K+-ATPase and basolateral AE2aredepolarizedand ex-pressed atreducedlevelsinthemurinechoroid plexus papil-loma which develops in mice transgenic for SV40 large T antigen.
Methods
Antibodies andreagents.Antipeptideantibodies to murine AE2 (28), tohumanankyrin (9),and toactin, myosin, a-actinin,andtubulin
(29)have beendescribed.Antibodyto seaurchin eggspectrin which cross-reactswith brainspectrin(fodrin)in manyspecies was from D. Begg(30). Additional antibodiesto human erythroidankyrin were
gifts ofV. Patel (31 ) and C. Cohen. Rabbit polyclonal antibodies to amino-terminal dodecapeptides of ratal (32) and ,BI (33) subunits of Na+K+-ATPase were raisedagainst HPLC-purified synthetic peptides coupled withsulfo-3-maleimidobenzoic acid N-hydroxy succinimide ester(PierceChemicalCo., Rockford,IL)via the COOH-terminal
cys-teinestokeyholelimpethemocyanin and thenaffinity-purifiedover
peptide-agarose columns (28). Secondary antibodies (Jackson Im-munoResearchLaboratories, Inc., West Grove, PA) included, for
im-munofluorescence, goat anti-rabbit and affinity-desorbed donkey
anti-rabbit antibodiescoupled to Texas red andaffinity-desorbed
don-keyanti-mousecoupled tofluorescein.Also used were goatanti-rabbit and goatanti-mousesecondaryantibodies coupled to alkaline
phos-phatase.
Bromochloroindolylphosphate and nitro blue tetrazolium were
from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD). LX-1 12 embedding resin was from Ladd Research Industries, Inc. (Burlington, VT). Other reagents were from Sigma Chemical Co. (St.
Louis, MO), Calbiochem-Novabiochem Corp. (La Jolla, CA), or
BoehringerMannheim Corp. (Indianapolis, IN).
SV40-TAg#188(8) mice transgenicfor SV40 enhancer region anti-gen,designatedTg(SV40E)Bri7, were provided by E. Sandgren and R. Brinster (34).
Tissue preparation. Rats and mice were anesthetized and perfu-sion-fixed asdescribed (35). Alternatively, animals were narcotized withCO2,andchoroid plexus was fresh-dissected into PBS or artificial CSF (36) with the aid ofadissecting microscope. The tissue was either
placeddirectly into SDS load buffer, or fixed in 3% paraformaldehyde,
quenched, and suspended in 3% agar-PBS whichwasallowed to solid-ify, and fixed for embedding (37). Transgenic mice were monitored for thedevelopment of ataxia and/or cerebellar bossing. Mice showing thesephysical signs were anesthetized, and choroid papilloma tumor andsurrounding choroid plexus were dissected as above.
Fertilized chickeggs atembryonicday 13weregiftsof Dr. D. Wu and Dr.C. Cepko.Choroid plexuswasfresh-dissected into artificial CSF and handled as above. Pig choroidplexuswasobtainedby dissec-tion ofheads obtained after surgery from the TuftsUniversitySchool of Veterinary Medicine(Medford, MA).
Transfections. p7OZN8 (22) encoding murine AE2 protein and pL2AAencodingmurinekidneyAE 1(38)weretransientlytransfected into COS-1 cellsusingthe DEAE-dextranprocedure(38, 39).
[35S]-Methionine labeling of transfected cells and immunoprecipitations
from wholetransfected cell lysates were performed as described (38, 39). Immunoblots. SDS-PAGE and electrophoretic transfer of protein tonitrocellulosewereperformedasdescribed(36).Antibodieswere usedatconcentrationsrangingbetween 1:50 and 1:500. Competitor
peptides were added to the incubations at concentrations between 4 and25tg/ml. Secondary antibodiescoupledtoalkalinephosphatase
wereused tolocalize antibodybinding, and blotsweredeveloped with
bromochloroindolyl phosphateandnitroblue tetrazolium. Proteinwas measuredby thebis-cinchoninic acidassay(PierceChemicalCo.).
Immunocytochemistry. The 3% agar blocks containingchoroid plexus werepostfixedat4°C foraweek,then embedded in LX-1 12 resin as described (37). Alternatively, small tissue fragments were
quick-frozen,freeze-dried,vacuum-embedded inEpon,andprocessed
forimmunostainingasdescribed(26). Semithinsections of nominal
0.5-1-,m thickness were cut on aSorvall Porter-Blum microtome.
Confocalmicroscopyof these sections(MolecularDynamics-Sarastro,
SanFrancisco, CA)indicatedtruethicknessesofupto2.5Am.
Anti-body incubations were modified from previously described proce-dures(36).
TransfectedCOS cells were plated onto 12-mmcoverslips,which werefixed in 3%paraformaldehyde,quenched,washed, mounted, and processed aspreviously described (39).
Results
Confirmation
that the band 3-related proteinofchoroidplexus is AE2. AE2 mRNAhasbeen detected in choroidplexus papil-loma(22) and normal choroid plexus, and thecDNAhas beenFigure 1. Immunopreci-pitationof AE2bythree anti-AE2peptide anti-bodies (asnoted) from a-CT a-ecto ca-cyto lysatesof AE2
cDNA-1224- 961- 426- transfected COS cells. P 1237 P 974 P 440 Thepredominant AE2
speciesis the 145-kD endoplasmic reticulum form(arrow). The
more
highly
processedGolgiform,barely
visi-_"
_
bleat- 160kD,wasnotprominentin these cells. Crude preimmune
sera(P)didnot
precipi-tateAE2from
trans-*
XPFe fected cell lysates.
14C--^
#;
Labeled molecular size standards inright-most_
~~~~~~~~~~lane
are (from bottom)~.,4~. 0 45,68,97, and(above
.,.-
~~~~~~~~~~~arrow)
--f ao 200 (bv200kD.
7 8 9 lO 11
<'- i
| | | :e". .5¢
i'
1
Figure 2. Specificity of immunoblot detection ofAE2inratchoroid plexus by three anti-AE2 peptide antibodies: Preimmuneserum(lane
1), crudeanti-424-440 (lane 2), affinity-purified anti-424-440 in
thepresenceof peptide antigen (lane 3), nonspecific peptide(lane
4),or nopeptide (lane 5);anti-961-974 in thepresenceof peptide
antigen (lane 6), nonspecific peptide (lane 7),or nopeptide (lane 8);anti-1224-1237 in thepresenceof peptide antigen (lane 9),
non-specific peptide (lane 10),ornopeptide (lane 11). Choroid plexus harvesteddirectly into SDS displayed onlyonematureAE2 bandof
165-kD Mr without degradation. Prestained size standards inthe rightmost lane above and below the AE2 bandare208and97kD.
cloned from choroid plexus. Furthermore, a band 3-related
polypeptide has been detected usingacross-reactiveantibody
tothecarboxy-terminal peptide of AE 1 (20). To establish
de-finitively the identity of this protein of the choroid plexus,we
developed antibodiestoAE2peptides and tested their specific-ity firstonCOS cells transiently transfected with AE2 cDNA
and thenonchoroidplexus. Fig. 1shows the results
ofimmuno-precipitation with affinity-purified immune or preimmune serafromdetergentlysates of
[31S]methionine-labeled
trans-fected COS cells. The core-glycosylated AE2 polypeptide of 145 kD(arrow)wasthepredominant biosynthetic product of thecells, with onlytracesofthe 165-kDmatureglycosylated AE2(38). The AE2 polypeptideswereprecipitated by all three
antipeptide antibodies but not by theircorresponding crude preimmunesera.Whereas anti- 1224-1237 precipitated kidney
AE1transiently expressed in COS cells, 96 1-974 and
anti-426-440,directed against unique AE2sequences,didnot
recog-nize AE1. Similarly, antibodies directed against unique AE1
sequences (36) and AE3 sequences (inpreparation) did not
recognize AE2 (not shown). Each of the three AE2 anti-bodies also detected transiently overexpressed AE2 in COS cellsby immunocytochemical staining (not shown).
The three antipeptide AE2 antibodieswere nextused to
search forAE2inchoroidplexus. Fig. 2 demonstrates specific immunodetection ofmatureglycosylated AE2 polypeptide in
ratchoroid plexus. The 165-kD
M,
of choroid plexus AE2wasof the samesizeas ingastricmucosa(28) andasthe(barely
visible)upperband of AE2-transfected COS cells (Fig. 1). The AE2band detected by each of the three antibodieswas
com-pletely abolished by inclusion of peptide antigen, butnot
non-specific peptide,intheantibody incubation mix.
When these antibodieswereused forimmunofluorescence
localization of AE2(Fig. 3, upperpanels), they each detected basolateralstaining ofthe choroid plexus epithelial cells.
Immu-Figure 3. Specificity of immu-nocytochemical detectionof AE2 inratchoroidplexus by three anti-AE2peptide anti-bodies. Each column
repre-sentssequentialsemithin
sec-tionsimmunostainedwith
an-tibodyalone(top row)orin
thepresenceofpeptide antigen
(middle row)ornonspecific peptide (bottom row). a,
anti-424-440; b, anti-1224-1237;
c,anti-961-974. Antibodiesto
allepitopesdetectedbasolateral
membranestaining. Bar=30
Am.
1432 Alper, Stuart-Tilley,Simmons,Brown,and Drenckhahn
nostaining was abolished by inclusion of peptide antigen (mid-dlepanels)but not nonspecific peptide (lower panels) inthe antibody incubation. Sequential sectionsincubated individu-ally with the three antibodies immunostained the basolateral membranesof identicalcells (not shown). Furthermore, anti-peptide antibodies raisedagainst two additional peptide epi-topes of AE2produced similarstaining patterns (not shown). In contrast, immunostaining of choroid plexus with isoform-specific antibody to AEI and to AE3 was negative (not shown).This colocalization of antigen-specific immunostain-ingbydistinct antibodiesagainst multiple peptide epitopes of AE2confirmed the identity ofthe band 3-related protein of choroid plexusasAE2.
The anti-1224-1237 antibody(Fig. 3, column b) consis-tently produced the strongest immunocytochemical signal among the antibodies tested, whetherused to detect AE2 in Eponsectionsof choroidplexus orgastricmucosa (28) or in transfected COS cells fixed on coverslips. Basal membrane stainingwasgenerally heavierthan was lateralstaining withall theantibodies.Inthe lateral membranesofchoroid plexus epi-thelial cells,immunostainingwas denser at the basal than at the apicalpoles.
Fig.4documents that all threeantipeptideantibodies also detectedmousechoroid plexus AE2 inanantigen-specific fash-ion (lanes 1-6).Inaddition,all threeantibodiescross-reacted withAE2inchoroid plexus from pig (lanes 7-9). Only two of theantibodies recognized AE2in choroidplexusfrom chick embryo (lanes 10-12). ThoughAE2 cDNA sequences from pigandchickhave not been reported, this immuno-cross-reac-tivity suggests considerable sequence conservation among mammals andbirds intherecognized epitopes.The cross-reac-tivity is lowest for anti-961-974,which recognizesthe poorly conservedlargeectoplasmicloop betweenputativemembrane spans 5 and 6ofAE2. Inthis region of AE2, sequence
diver-1 2 3 4 5 6 7
Figure5. Immunoblotcharacterizationof choroidplexus
Na'K'-ATPasef#1(lane1)andalIsubunits(lane2);ankyrinfrom
choroidplexus (lane 3)andfromwhole blood(lane4);ankyrinfrom
unperfused choroidplexus (lane 5)andperfused,bloodcell-free choroidplexus (lane 6);andchoroi plexusforin(lae 7).
1 2 3 4 5 6
208
-I
97---0.t
68
-
45-45 - ,
7 8 9 10 1112
W .S~~~~~~~~~I
Figure 4. Immunoblot reactivity across species by anti-AE2 peptide antibodies(arrow).Mousechoroid plexus AE2 detected by
anti-1224-1237 (lane 1), anti-961-974 (lane 3), andanti-424-440(lane
5),orin the presence of therespective peptide antigens(lanes 2, 4, and6);Pigchoroid plexus AE2 detected by anti-1224-1237 (lane
7), anti-961-974(lane8),andanti-424-440 (lane 9); Chickchoroid
plexus AE2 detected byanti-1224-1237(lane10) and anti-424-440 (lane 12). Anti-961-974 (lane 11) failed to cross-react with chick AE2.Mrof prestained size standards indicated atleft.
Figure6. Immunoloca-lizationinsequential semithin LX-112
sec-tions ofratchoroid plexus ofAE2(a),aI Na+K+-ATPase(b),
andfodrin(c).Bar=30
Am.
gence between mouse and chick exceeds the cross-reactivity of anti-mouseantibody.
Colocalization ofapical Na+K+-ATPasewith ankyrin and thespectrin/actin cytoskeleton. Fig. 5 documents the specific-ity in rat choroid plexus of antibodies raised against
Na'K+-ATPase,ankyrin, and nonerythroid spectrin. Antibod-ies to ratf1 and a1 subunits of Na+K+-ATPase detected, re-spectively, the expected broad band at - 55 kD(lane 1) and
the narrower band at- 100 kD (lane2). The a1 doublet band
intensities were specifically attenuated by peptide antigen. Both bands appeared not only with this particular antibody to theNH2-terminal 12aminoacids,but alsowith McKl mono-clonalantibody( 17) and with three other polyclonal antisera (not shown). Evaluation ofimmunoreactive ankyrin in cho-roid plexus was potentially problematic, since chocho-roid plexus is ahighlyvasculartissue,andcontaminatingerythrocytes could
provide apositive immunoblotsignal. Ankyrin from erythro-cytes(lane 3) andfrom choroidplexus(lane 4) comigrated at
- 220 kD.Semithin sectionsofchoroidplexusfrom the
per-fused rats were devoid of erythrocytes (not shown).Therefore, ankyrin immunoblots of erythrocytes andchoroidplexus from paired,nonperfused (lane 5), andperfusedrats(lane 6) were compared, and demonstrated that choroid plexus epithelial
cells expressed endogenousankyrinoranimmunoreactive iso-form.Antibodyto seaurchin spectrinwhich has broad
reactiv-ity across phyla (30) detected aprincipal band of - 240-260
kD(lane 7).
Theseantibodies were applied as immunocytochemical re-agents to sequential semithin sections ofrat choroid plexus
(Figs. 6-8). Unlike the basolateral localization of AE2, Na+ K+ -ATPasewasrestrictedtotheapicalcellsurface. Immu-noreactiveforms ofspectrin(fodrin), ankyrin,andactin were also concentratedapicallyandcolocalized with
Na+K+-ATP-ase(Figs.7and8).As in other
transporting epithelial cells,
thedistribution of
spectrin
and actin was notstrictly polarized.
Bothproteinswerealsoobserved
along
the basolateral aspects of theseepithelial cells,albeit withapparently
lowerconcentra-tions thanapically.However, unlike the situation in other sim-pletransportingepithelial cells with a free, nonattached apical surface, Na+K+-ATPase was clearly restricted to the apical membrane and absentfrom the basolateral surface. Ankyrin immunostaining, always less prominent than spectrin staining, was minimal orabsent at thebasolateral membrane. Similar observations were obtained with sections ofhuman choroid plexus(not shown).
This striking reversal of the distribution of the Na+K+-ATPase-ankyrin complex (and of the expression of other basolateral proteins, see Introduction) in the choroid
plexusraisedthequestion ofwhethermicrotubules might be
causallyinvolved ingenerationormaintenance ofthis phenom-enon.Recentstudiesonmicrotubulesin theintestinal epithe-liumandkidneytubulesindicateda roleofmicrotubules in the vectorial transportof membrane proteins to the apical mem-brane domain (26, 27, 40). Disruption of microtubules in thesepolarized epithelialcells causedredistribution of several apicalmembraneproteinstothe basolateral cellsurfaceor to intracellular vacuoles, whereas the basolateral localization of the Na+K+-ATPaseremainedunaffected inthese cells (26 and Alper,S. L.,unpublished observations).Asshown inFig. 9 A, microtubules were labeledthroughout the plexus epithelium, with aslight apical orientation. 6 h after intraperitoneal admin-istrationof colchicine(Fig. 9 B), mostoftheintracellular
im-munostaining of tubulinhaddisappeared,and only theapical stainingremainedatreduced levels.Thisindicatesthat the
or-ganizationalcenter for microtubules in choroid plexus is lo-cated beneath theapicalcellsurface,asisthecasein the
intes-tinalepithelium (26)and in MDCK cells (41).Incontrast to
apical membraneproteinsinintestinaland kidney epithelia,
the distribution ofNa+K+-ATPase withinthe apical mem-brane of the choroid plexus was not affected by colchicine treatment.Alsounaffectedwerethesubcellulardistributions of spectrin (fodrin), actin, andankyrin.Basolateral AE2 of the choroid plexus was similarly unperturbed in its distribution (notshown). Thus, themicrotubulesdonotappeartobe
im-re 7.Immunocytochemical lo-calization ofankyrininratchoroid
plexusbyantibodyraisedagainst
humanerythroid ankyrin.The
6-gum
frozen section showsankyrin
im-munoreactivitynotonlyatthe api-calenithelial cell membrane but also in membranes oferythrocytesof the plexuscapillaries.Bar= 10,um.
Figure 8. Nonsequential
6-Am frozensections of
ratchoroidplexus
im-munostainedwith anti-bodies toactin (A),
myosin(B), and a-ac-tinin(C).Allpanels showpredominantly apicalstaining.Bar=20 z =_ w
,u~~~~~m.
portant for generation and/or maintenance ofthe polarized distribution of Na+K+-ATPase and ofAE2 in the choroid plexus.
Another structure that has been suggested to be involved in the controlof the exocytotic incorporation ofmembrane pro-teins into the apicalplasma membrane is the apical terminal web(26, 42).The majorconstituents of this web are the actin
cross-linkingproteins spectrin(fodrin), a-actinin,andmyosin
(43). Asshownin Figs. 6-8, all fourcytoskeletal proteins are enriched underneaththeapicalplasma membrane. This
indi-catesthat thechoroidplexuscontainsawell-developed apical terminalwebwhichobviously does not constitute an effective barriertothe accessofNa+K+-ATPase-containing exocytotic vesiclesto the apical plasma membrane.
We examined the fate ofapical and basolateral intrinsic membrane transport proteins in another setting in which polar-ity is frequently disrupted, namely malignancy. We took ad-vantageof the propensity of mice transgenic for theSV40large
T antigen driven by its own promoter to develop choroid plexus papilloma between3and 5moofage(35). Inan earlier study, the papilloma expressed substantial amounts ofAE2 mRNA (22). However, when semithin sections were exam-ined for AE2immunostaining, AE2wasabsent frommost vi-sual fields(Fig. 10, b and c), and when (rarely) present was expressed circumferentially in the morphologically depolar-izedtumor cells(Fig. 10 a). Cross-reactiveAE 1in erythrocytes wasbrightly stained (Fig. 10, a-c). Na+K+-ATPase al sub-unit wassimilarlydepolarizedbut in most visual fields was still well abovethe threshold forimmunocytochemical detection (Fig. 10,d-f).
Discussion
The abundant expression of basolateral AE2 and apical Na+K+-ATPase in choroid plexus epithelium suggests that both playa prominent role in the secretion of CSF. The api-cally situated ATPase probably contributes to the decreased CSF
[K+].
Basolateral AE2(orAEl)inother tissues(28,36, 44) is generally associated with transepithelial transport of acid-baseequivalents. Since the principal function of the cho-roid plexus is thetransepithelialtransportofNaCl,NaHCO3, andwater,along with nutrients and secretedproteins,itseems reasonable to hypothesize the presence in choroid plexus of basolateralNa/H exchangeaswell. Sinceour attempts to de-tectNa+/H+exchanger 1 by immunoblot and immunocyto-chemistrywereunsuccessful,wesuggestthepresenceofan al-ternateNa+/H+ exchangerisoform, thoughother basolateral Na+entryways mayalsoexist. Further studiesonCSF elabora-tion would be facilitated by development of polarized choroid plexus cells in primary culture and eventually in permanent culture.Thelocalization of ankyrintotheapical membraneaway from AE2suggeststhattheankyrin isoformsdetectable with antibodiestoerythroid ankyrindonotbindtoAE2. Thesame conclusion issues from the localization of AE2tothe basolat-eral membrane(28) and immunoreactiveankyrintothe api-covesicular membrane ( 10) oftheparietal cell. Attempts to localize the ankyrin-bindingsiteon AE 1 haveconcluded that thebinding interfaceextendsover> 100amino acidsandmay also involve the far amino terminus(45). Thus, the several short regionsofhighlyconservedsequencewithin the
amino-terminalcytoplasmicdomains ofAE1,AE2,andAE3(which
do not include the very
divergent
far amino-terminalse-quences)probablydonotcomprisea commonbindingsite for asingle ankyrinisoform.Itremainspossiblethat alternate
an-kyrinisoforms suchasANK2orANK3(45)mayinteract with AE2, but suchanankyrinmightnotbindtothea1 subunit of theNa+K+-ATPase. ChoroidplexusankyrincDNAsequences havenotbeenreported.
Thedegreetowhichthechoroidplexusis trulyan
epithe-liumofreversedpolarity isunresolved.Clearly,someaspects of polarized secretion and membrane protein insertion are
re-versed compared with renal and intestinal epithelial cells. Thoughapical Na+K+-ATPase in one cloneofMDCK cells (4, 8) isenzymatically inactive,thisappearsnot tobetrueof theapicalATPaseofthechoroid plexus.Apicalsphingolipids
inMDCKcells have beenpostulatedtoinactivatethatATPase, but theapicallipid composition of choroid plexus has not been reported. The retinal pigment epithelial cell also colocalizes Na+K+-ATPaseandankyrintoits apical membrane,but that
Figure 9. Ratchoroidplexus immunostained with antitubulin antibody before (A) or5hafter (B) administration of
intra-peritoneal colchicineasdescribed(23). B shows retention in theepithelialcellsonlyofsome apicalmembrane-associated
tubulin immunoreactivity.Bar=20 ym.
steadystatedistribution isdependentonapical surfacecontact with the photoreceptor cellswhichoverlieit insitu.Other
indi-cesofpolarity,suchasviralbudding,haveyet tobereported for choroidplexus.
Onepossible mechanismresponsible forthepartialreversal ofpolarityinchoroidplexus could beareversalofthe
orienta-tion and polarity ofthe microtubule system. In enterocytes, proximal tubularepitheliumofthekidney,andMDCKcells, microtubules have been shownto beimportant for vectorial
deliveryof membrane proteinsto theapical cellsurface (26, 27,40, 41). The structural basis for vectorial transport from theGolgiapparatus totheapicalplasma membrane isprovided
in part bytheuniform orientation andalignment of
microtu-bules. Thesehavetheirorganizingcentersunderneath the
api-calterminalweb(subterminal space).Asshowninthis study, this general pattern of microtubule orientation appears alsoto bepresent inthechoroid plexusepithelium.However, disrup-tion of themicrotubule system by colchicine didnot haveany detectableeffect on the apical position of Na+K+-ATPaseor onthe basolaterallocalizationofAE2.This indicatesthat mi-crotubules areprobably notinvolvedin thepartial reversalof polarity in these cells.Inintestineandkidney,basolateral tar-geting of Na+K+-ATPase and ofa 120-kDmembraneprotein was not perturbed by colchicine, whereas both apical and baso-lateral vacuolar H+ -ATPase ofrenal intercalated cells were perturbed (46). Thus,thedisruptive effects of colchicineare not surface specific, but rather are protein specific. Another
possible interpretation might be that anyproteins, apical or basolateral,which bindviaankyrintothemembrane
cytoskele-ton areresistanttodelocalizationbycolchicine.
Asecondmechanism for partialreversalof polarityin the
choroid plexusmight be theterminalweb,which in exocrine
glandsplaysarole in thecontrolof apicalexocytosis (3, 42). If thechoroidplexusepitheliumlacked anapical terminalweb, one could speculate that the loss ofthis exocytotic barrier would facilitate apical incorporation ofbasolateral proteins. Butsinceactinand theprincipal actin cross-linking proteins of
the terminal web (a-actinin, myosin, and spectrin) are enriched underneath theapicalcellsurfaceoftheplexus
epithe-lium, the terminal webappears tobe welldeveloped in these cells.Therefore,analtered terminalwebstructure cannot serve toexplainthepartial reversal of polarityinthe choroid plexus. Athird cytoskeletal mechanism that is believed to be in-volved in placement ofan ever-growing number ofintegral membraneproteins (i.e., Na+K+-ATPase and voltage-gated Na+channel)tospecialized sites ofthe cell surface is linkage of theseproteins via ankyrin to thespectrin/actin-based mem-brane cytoskeleton (seeIntroduction). This mechanism may also operate in the choroid plexus epithelium, where Na+K+-ATPaseandimmunoreactive ankyrinarecolocalized attheapical cell surface andare absentfrom the basolateral membranedomain. If Na+K+-ATPase is delivered ina non-polarized fashiontoboth theapicalandbasolateralsurfaces (4, 8, 39), onlytheapical fraction ofNa+K+-ATPase will be
Figure 10. Selected fieldsof semithin sections
fromchoroidplexus papilloma removedfrom
anunperfusedmousetransgenic for SV40large TantigendrivenbytheSV40 early promoter-enhancerimmunostainedwithantibodiesto AE2 (a-c) and to Na+K+-ATPase al subunit (d-f).Bar= IOAm.
lized via linkagetoapical ankyrintotheunderlyingmembrane cytoskeleton.In contrast,moleculesinserted basolaterallymay berapidly endocytosed and either degradedordeliveredtothe
apicalcell surface bytranscytosis.Suchatranscytotic pathway hasbeen well established for the poly-Ig receptor (47) and variousFcreceptors (48)in polarizedmonolayers, as well as for hepatocyte proteins ofthe biliary canalicular membrane in vivo(49).Directapicaltargetingis alsopossible.
However,noneof theseexplanationsfor the apical localiza-tion of Na+K+-ATPase easily explains theapicalenrichment ofankyrin in the choroidplexus.E-cadherin hasbeen shown to bedirectlyorindirectlyassociated withankyrinandmayplaya roleinstabilizingandassemblingthe membranecytoskeleton and the Na+K+-ATPase at the basolateral cell surface of MDCK cells(50).Recentstudies indicate that chick choroid plexus epithelial cells express B-cadherin (51) which, when transfected into fibroblasts, failsto condenseendogenous fo-drin and Na+K+-ATPase at thelateralmembrane.This behav-iorcontrasts with thatof E-cadherin whichinfibroblasts(52) and in retinal pigment epithelial cells (53)condenses fodrin and Na+K+-ATPase at the lateral membrane. Thus, the ab-senceof cadherins capable ofassemblingATPase-cytoskeleton complexesatthebasolateral cell surface ofthechoroid plexus couldcontributetoaccumulation ofankyrin atthe apical sur-face. However, the possibility remains of apical membrane
binding sites for choroid plexus ankyrin in addition to the Na+ K+ -ATPaseitself.These couldincludeone ofthe
ankyrin-binding glycoproteins(54). Tofurthertestthis hypothesis,as well as to assess protein-sorting mechanisms in the choroid plexus,wewillneed anin vitrosystem in which to study devel-opmentof polarityunder controlled conditions.
Acknowledgments
We thank Dr. Seymour Rosen for use of themicrotome,Dr.James Nathanson fordiscussionanddemonstration,Dr.DouglasRobinson fordissection ofpig choroid plexus, and Dr.Eric Sandgrenand Dr. Ralph Brinsterfortransgenicmice.
This workwassupported by National Institutes of Health grant DK-43495 and a grantfrom theCystic Fibrosis Foundation (S. L. Alper).
References
1.Rodriguez-Boulan, E., and S. K. Powell. 1992.Polarityofepithelialand neuronal cells.Annu. Rev.Cell Biol. 8:395-427.
2.Bomsel, M., andK. Mostov. 1992. Role ofheterotrimericGproteinsin membrane traffic. Mol. Biol.Cell.3:1317-1328.
3. Rothman,J.E.,and L.Orci. 1992. Molecular dissection ofthesecretory
pathway. Nature(Lond.).355:409-415.
4.Hammerton, R. W.,K. A.Krzeminski,R. W.Mays,T. A.Ryan,D. A.
Wollner, andW. J.Nelson. 1991. Mechanism forregulatingcell surface
distribu-PolarityofCytoskeletonandIonTransportersinChoroidPlexus 1437
tion ofNa',K+-ATPase in polarized epithelial cells. Science(Wash. DC).
254:847-850.
5.Drenckhahn, D., K.Schluter, D. P. Allen, and V. Bennett. 1985.
Colocali-zation ofband 3 withankyrinandspectrinatthe basal membrane of intercalated cellsinthe ratkidney. Science(Wash. DC). 230:1287-1289.
6. Koob, R., M.Zimmermann,W.Schoner,and D. Drenckhahn.1987. Colo-calizationandcoprecipitationofankyrinandNa',K+-ATPaseinkidney
epithe-lial cells. Eur. J.Cell Biol.45:230-237.
7. Koob, R., D.Kraemer, G.Trippe,U.Aebi, and D. Drenckhahn. 1990.
Association of kidney andparotidNa',K+-ATPasemicrosomeswithactin and
analogsofspectrin andankyrin.Eur. J.Cell Biol. 53:93-100.
8.Zurzolo,C.,E. Rodriguez-Boulan, C. Gottardi,M. J.Caplan, K. A.K.
Siemers,R. H.Wilson,R. W.Mays,T. A.Ryan,D. A.Wollner,and W.J. Nelson.
1993.DeliveryofNa+K+-ATPasein polarized epithelialcells.Science(Wash.
DC).260:550-556.
9. Drenckhahn, D., and V. Bennett. 1987. Polarized distribution ofM,
210,000 and 190,000 analogsoferythrocyte ankyrin along the plasma membrane
oftransporting epithelia,neuronsandphotoreceptors. Eur. J.Cell Biol.
43:479-486.
10.Smith,P., A. L.Bradford,E.-H.Joe, K. J.Angelides,D. J. Benos, andG. Saccomani. 1993. Gastricparietal cell H',K+-ATPase microsomesare
asso-ciated withisoforms of ankyrin and spectrin.Am.J.Physiol. 264:C63-C70.
I1.Gundersen, D., J.Orlowski,and E.Rodriguez-Boulan. 1991. Apical polar-ity ofNa,K-ATPaseinretinal pigment epithelium is linkedto a reversalofthe
ankyrin-fodrin submembrane cytoskeleton. J. Cell Biol. 112:863-872.
12. Gundersen, D.,S.K.Powell, and E. Rodriguez-Boulan. 1993. Apical
polarization ofN-CAM inretinalpigmentepitheliumisdependentoncontact with the neuralretina.J.Cell Biol.121:335-343.
13. Cole,T., P. W.Dickson,F.Esnard, S.Averill,G. P.Risbridger,F.
Gauth-ier,andG.Schreiber. 1987. ThecDNA structure andexpressionanalysisofthe genesforthecysteine proteinase inhibitor cystatinC andforbeta2-microglobulin
in ratbrain. Eur. J.Biochem. 186:35-42.
14. Aldred, A. R., P. W.Dickson, P.D.Marley, and G.Schreiber. 1987.
Distribution of transferrin synthesisinbrainand othertissuesin the rat. J.Biol. Chem. 262:5293-5297.
15.Aldred, A. R., A. Grimes,G.Schreiber, and J. F. Mercer. 1987.Rat
ceruloplasmin. Molecularcloningandgeneexpressioninliver,choroid plexus, yolksac,placenta,andtestis. J.Biol.Chem. 262:2875-2878.
16.Duan,W.,T.Cole, and G. Schreiber. 1989. Cloningandnucleotide
se-quencing oftransthyretin (prealbumin) cDNA fromratchoroid plexusandliver. Nucleic AcidsRes. 17:3979.
17. Sweadner, K. W. 1989. Isozymes ofthe Na+K+ATPase. Biochim. Biophys.Acta.988:185-220.
18.Zlokovic,B.V., J. B.Mackic,L.Wang,J.G.Mccomb,andA.
McDon-ough. 1993. Differentialexpression of Na,K-ATPaseasubunitand#subunit isoforms attheblood-brain barrier and thechoroid plexus. J. Biol. Chem. 268:8019-8025.
19. Watts, A.G., G. Sanchez-Watts,J. R.Emanuel,and R. Levenson. 1991.
Cell-specific expressionofmRNAsencoding Na+K+-ATPase alpha-and beta-subunit isoforms within theratcentralnervoussystem. Proc.Natl. Acad. Sci. USA.88:7425-7429.
20.Lindsey,A.E.,K.Schneider,D. M.Simmons,R.Baron,B.S. Lee,and R. R. Kopito. 1990.Functional expressionandsubcellular localization ofan
anion exchangercloned from choroid plexus. Proc. Natl. Acad. Sci. USA.
87:5278-5282.
21. Alper, S. L., A.Stuart-Tilley, andD. Brown. 1991.Tissue distribution of
theCl-/HC03 exchanger AE2:abundantexpressionin thechoroid plexus.
Kid-ney Int.38:71la.(Abstr.)
22.Alper,S. L., R. R.Kopito,S. M.Libresco,and H. F.Lodish.1988.Cloning
andcharacterization ofamurineband 3-related cDNAfromkidneyandfroma
lymphoidcellline.J.Biol.Chem. 263:17092-17099.
23. Alper,S. L. 1991.The band 3-related anionexchanger (AE)genefamily.
Annu.Rev.Physiol.53:549-564.
24.Ernst, S. A., J.R.Palacios,andG.J.Siegel. 1986. Immunocytochemical localizationofNa+K+-ATPasecatalytic polypeptideinmousechoroid plexus.J.
Histochem.Cytochem.34:189-195.
25.Saito,Y., and E.M.Wright. 1983.Bicarbonate transport across thefrog choroid plexusandits controlbycyclicnucleotides. J.Physiol.336:635-648.
26.Achler,C., D. Filmer, C. Merte, andD.Drenckhahn. 1989. Roleof micro-tubules inpolarized deliveryofapicalmembraneproteinstothe brush borderof
theintestinal epithelium.J.Cell Biol.109:179-189.
27.Gutmann, E. J., J. L. Niles,R. T. McCluskey, and D. Brown. 1989. Colchicine-inducedredistributionofanapicalmembraneglycoprotein(gp330)
inproximaltubules. Am. J.Physiol.257:C397-C407.
28.Stuart-Tilley, A., C. Sardet, J. Pouyssegur, D. Brown, and S. L. Alper. 1994.Immunolocalization ofanionexchanger AE2 and cation exchangerNHE1
indistinct, adjacent cells of gastric mucosa. Am. J.Physiol.Inpress.
29. Drenckhahn, D., and R. Dermietzel. 1988. Organizationof the actin
filamentcytoskeletonin theintestinalbrush border.Aquantitativeand
qualita-tive immunoelectronmicroscopestudy.J.CellBio. 107:1037-1048.
30.Fishkind,D.J., E. M. Bonder, and D. A. Begg. 1990. Subcellular
localiza-tion ofseaurchineggspectrin: evidence for assembly ofthe membrane-skeleton onuniqueclassesof vesiclesin eggs andembryos. Dev. Biol. 142:439-452.
31. Patel, V. P.,andH. F.Lodish.1987. Afibronectinmatrix is required for
differentiationof murineerythroleukemiacells intoreticulocytes. J. CellBiol. 105:3105-3118.
32. Shull, G.,J.Greeb,and J. B.Lingrel. 1986. Molecular cloning ofthree
distinct forms of theNa',K+-ATPasea-subunit fromratbrain.Biochemistry. 25:8125-8132.
33.Mercer, R. W., J. W.Schneider,A.Savitz,J.Emanuel,E. J. Benz, and R. Levenson. 1986.Rat-brain Na,K-ATPase beta-chaingene:primarystructure,
tissue-specific expression,andamplificationinouabain-resistantHeLaC+ cells. Mol.Cell Biol. 6:3884-3890.
34. Palmiter,R.D., H. Y. Chen, A.Messing,and R. L.Brinster.1985.SV40 enhancerand large-Tantigenareinstrumentalin development ofchoroid plexus
tumoursintransgenic mice.Nature(Lond.).316:457-460.
35. Alper, S. L., J.Natale, S.Gluck,H.F.Lodish,and D. Brown. 1989. Definitionofintercalated cell subtypes inratkidneycollectingductusing antibod-iesagainst erythroid band 3and renalvacuolarH'ATPase. Proc.Nail. Acad. Sci. USA. 86:5429-5433.
36. Nathanson, J.A.,andL. L.Chun.1989.Immunological function of the blood-cerebrospinalfluid barrier.Proc.Natl.Acad. Sci. USA. 86:1684-1688.
37.Ercolani, L.,D.Brown,A.Stuart-Tilley, and S. L. Alper. 1992. Colocali-zationof GAPDH and band 3 (AEI)proteinsinraterythrocytes andkidney intercalated cell membranes.Am.J.Physiol. (Cell).262:F892-F896.
38. Alper, S. L.,F.C.Brosius,A. M.Garcia, S. Gluck,D. Brown, and H. F.
Lodish. 1989.Two geneproductsencoding putativeanionexchangers ofthe
kidney.InAnion Transport Proteins oftheRed Blood CellMembrane,Kidney,
andDiverseCells. N. Hamaskiand M. L.Jennings,editors. Elsevier Science PublishersB.V.,Amsterdam. 153-164.
39.Haller,C.,and S. L.Alper. 1993.Nonpolarizedsurface distribution and delivery ofhuman CD7 inpolarizedMDCK cells. Am.J.Physiol. 265:C1069-C1079.
40.Eilers, U.,J.Klumperman,and H.-P.Hauri. 1989. Nocodazole,a micro-tubule-activedrug, interferes withapical protein deliveryin culturedintestinal epithelialcells(Caco-2).J.CellBiol. 108:13-22.
41.Bacallao, R., C.Antony,C.Dotti,E.Karsenti, E. H.K.Stelzer, and K. Simons. 1989.ThesubcellularorganizationofMadin-Darbycaninekidneycells during formation ofapolarizedepithelium.J.CellBiol. 109:2817-2832.
42.Aunis, D.,and M.-F.Bader. 1988.Thecytoskeletonas abarrierto
exocy-tosis insecretory cells.J.Exp. Biol. 139:253-266.
43.Drenckhahn, D.,andH.-G. Mannherz. 1983. Distribution of actin and actin-associatedproteins myosin, tropomyosin, a-actinin, vinculin,andvillin in
ratandbovine exocrine glands.Eur.J.CellBiol.30:167-176.
44.Home, W., M.Moya,L.Neff,S.Kellokumpu,R.Kopito, S. Alper,and R. Baron. 1991. Aband3-relatedchloride/bicarbonate exchanger is highly ex-pressed at thebasolateralmembraneofosteoclasts.J. Bone Miner. Res.6:S57.
45.Lambert,S.,and V. Bennett. 1993. Fromanemiatocerebellar dysfunc-tion:areview oftheankyringenefamily.Eur. J.Biochem. 211:1-6.
46.Brown, D.,I.Sabolic,and S.Gluck.1991.Colchicine-induced redistribu-tionofproton pumpsinkidney epithelialcells.KidneyInt.33(Suppl.):S79-S83.
47.Apodaca,G.,M.Bomsel,J.Arden,P.P.Breitfeld,K.Tang, andK. E. Mostov. 1991. Thepolymeric immunoglobulin receptor. A modelproteinto
studytranscytosis. J. Clin.Invest.87:1877-1882.
48.Hunziker, W.,and I. Mellman.1989.Expression
ofmacrophage/lympho-cyte Fc receptors inMadin-Darbycanine kidneycells.Polarityandtranscytosis differfor isoforms withorwithout coatedpitlocalization domains.J.Cell Biol.
109:3291-3302.
49.Bartles,J.R.,H. M.Feracci,B.Steiger,andA.L.Hubbard. 1987.
Biogen-esisofthe rathepatocyteplasmamembrane invivo.Comparisonofthe pathways
takenbyapicaland basolateralproteinsusing subcellular fractionation.J.Cell Biol. 105:1241-1251.
50.Nelson,W.J.,E. M.Shore,A. Z.Wang, andR.W. Hammerton.1990.
Identification of amembrane-cytoskeletal complex containingthecell adhesion moleculeuvomorulin (E-cadherin), ankyrin,andfodrininMadin-Darbycanine
kidney epithelialcells.J. Cell Biol.110:349-357.
51.Mars,J. A., and W. J. Nelson. 1993.Membrane-cytoskeleton,B-cadherin,
andapical Na+,K+-ATPase polarityin thechoroidplexusepithelium. J.Cell. Biochem.Suppl. 17B:27Ia.(Abstr.)
52.McNeill,H., M.Ozawa,R.Kemler,and W. J. Nelson. 1990. Novel func-tionofthe celladhesionmoleculeuvomorulinas aninducer ofcellsurface polar-ity. Cell.62:309-316.
53.Andersson-Fisone, C.,I.Nabi,A.P.Mathews, C.Zurzolo,andE.
Rodri-guez-Boulan.1993. LaterallocalizationofNa,K-ATPaseinE-cadherin(L-CAM)
transfected rat retinalpigment epithelialcells(RPE).J.Cell. Biochem.Suppl.
17B:270a. (Abstr.)
54. Davis,J.Q.,T. McLaughlin,and V.Bennett. 1993.Ankyrin-binding proteinsrelatedto nervoussystem cell adhesion molecules.Candidatestoprovide
transmembrane and intercellular connections in adult brain. J. Cell Biol.
121:121-133.
