Plakophilin, armadillo repeats, and nuclear localization

Plakophilin, Armadillo Repeats, and Nuclear LocalizationMICHAEL W. KLYMKOWSKY*Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347

ABSTRACT Plakophilins are armadillo-repeat containing proteins, identified through theirlocalization to desmosomes. Expressed in a wide range of tissues, plakophilins are largely nuclear inmost cell types [Schmidt et al. (1997) Cell Tissue Res 290:481; Mertens et al. (1996) J Cell Biol135:1009]. Using Xenopus embryos and cultured A6 cells, together with myc- and green fluorescentprotein (GFP)-tags, we found that both the N-terminal, non-armadillo repeat ‘‘head’’ and theC-terminal armadillo repeat-containing regions can enter nuclei. The ‘‘arm’’ repeat domain ispredominantly cytoplasmic and concentrated at the cell cortex, whereas the head and full-lengthpolypeptides are concentrated in the nucleus. The head domain can also be seen to decorate anddisrupt keratin filament network organization in some cells. In the course of these studies, we foundthat the distribution of the myc-epitope and green fluorescence differed in fixed cells, e.g., while thegreen fluorescence of a myc- and GFP-tagged head domain polypeptide was usually exclusivelynuclear, a substantial fraction of the myc-immunoreactivity was cytoplasmic. Treating cells with thetranslation inhibitor cycloheximide reduces the cytoplasmic myc-signal, suggesting that it repre-sented nascent polypeptides awaiting folding and nuclear import. Based on these types ofexperiments, GFP can be seen as a marker of the distribution of the mature form of the taggedpolypeptide. Microsc. Res. Tech. 45:43–54, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTIONWhere proteins actually are in cells, and where they

function, can be difficult questions to answer. Proteinsonce thought of as exclusively cytoplasmic, such as thecadherin-binding proteins b-catenin (Fagotto and Gum-biner, 1994; Schneider et al., 1996; Yost et al., 1996),plakoglobin (g-catenin) (Karnovsky and Klymkowsky,1995; Rubenstein et al., 1997), plakophilins-1 (Schmidtet al., 1997) and -2 (Mertens et al., 1996), the tightjunction-associated proteins symplekin (Keon et al.,1996) and ZO1 (Gottardi et al., 1996), and the adenoma-tous polyposis coli tumor suppressor protein (Neufeldand White, 1997) have also been found localized tonuclei. At same time, there is a growing realization thatproteins can migrate dramatically, both into and out ofthe nucleus, during the course of fixation and subse-quent immunocytochemical staining (see Melan andSluder, 1992; Mertens et al., 1996).

An informative example of this problem is providedby the Drosophila segmental polarity gene productarmadillo1 and its vertebrate homologs b -catenin andplakoglobin (g-catenin). These are the defining mem-bers of the arm-repeat family of proteins, which in-cludes other cadherin-binding proteins (see below) andthe nuclear localization sequence (NLS) receptor pro-teins karyopherin-a /importin-a and b (see Malik et al.,1997). Originally identified through mutations thatdisrupt the establishment of embryonic segment polar-ity (Wieschaus and Riggleman 1987), immunocyto-chemical studies indicated the protein was associatedwith the cellular periphery and accumulated cytoplas-mically in regions of the embryo subject to wingless(wnt) signaling (Peifer and Wieschaus 1990; Rigglemanet al., 1990). While genetic studies indicated that thearmadillo1 gene product was involved in the regula-

tion of other genes, it was originally not found withinnuclei (see Peifer and Wieschaus, 1990; Riggleman etal., 1990). Subsequent studies on the vertebrate ho-mologs of armadillo, b-catenin, and plakoglobin re-vealed the nuclear localization of these proteins (seeFagotto and Gumbiner, 1994; Funayama et al. 1995;Karnovsky and Klymkowsky, 1995; Rubinstein et al.,1997; Schneider et al. 1996; Yost et al., 1996). InDrosophila, the nuclear localization of armadillo can beseen best using mutant forms of the protein that nolonger associate with cellular adhesion junctions (seeFigs. 4 and 5 in Orsulic and Peifer, 1996).

Armadillo, b-catenin, and plakoglobin are character-ized by a central stretch of 12 ‘‘arm’’ repeats (Peifer etal., 1994; Riggleman et al., 1989), the structure of whichhas recently been determined for b-catenin (Huber etal., 1997) and karyopherin-a (Conti et al., 1998). Muta-tional analysis of b-catenin and plakoglobin indicatesthat it is their central arm repeat regions that arerequired for nuclear localization (Funayama et al.,1995; Karnovsky and Klymkowsky 1995; Rubenstein etal., 1997). Sequence analysis, however, fails to identifyan obvious nuclear localization sequence (NLS) in thisregion and in a cell-free system b-catenin is importedinto nuclei in the absence of classical NLS-receptors,i.e., karyopherins/importins (Fagotto et al., 1998). TheseNLS receptors are themselves armadillo-repeat pro-teins (Conti et al., 1998; Malik et al., 1997), suggestingthat the presence of armadillo repeats may be sufficientto drive nuclear import.

To study this latter question, we analyzed the nuclearlocalization of the desmosomal protein plakophilin-1

1Genes appear in italics, proteins in plain text.

Contract grant sponsor: NIH; Contract grant number: GM54001.*Correspondence to: Michael W. Klymkowsky, Molecular, Cellular and Develop-

mental Biology, University of Colorado, Boulder, Colorado 80309–0347.E-mail: [email protected]

Received 24 August 1998; accepted in revised form 19 October 1998

MICROSCOPY RESEARCH AND TECHNIQUE 45:43–54 (1999)

r 1999 WILEY-LISS, INC.

(Hatzfeld et al., 1994; Heid et al., 1994). Plakophilin-1aassociates with epidermal desmosomal cadherins andis localized to nuclei, whereas a splice variant, pla-kophilin-1b, which contains an insert of 21 amino acids,is found exclusively in nuclei (Schmidt et al. 1997).Plakophilin-2, a closely related protein, is also nuclearin a wide range of cells (Mertens et al., 1996). Sequenceanalysis indicates that the plakophilins are related toarmadillo, b-catenin, and plakoglobin (Hatzfeld, 1997),forming a distinct subfamily that includes the classicalcadherin-binding protein p120ctn (Reynolds and Daniel,1997), p100 (a cadherin-binding protein related top120ctn) (Staddon et al., 1995), p0071 (Hatzfeld andNachtsheim, 1996) and the related protein p6542 (seeHatzfeld, 1997), the armadillo repeat gene deleted inVelo-cardio-facial syndrome (ARVCF) protein (Sirotkinet al., 1997), and the neural, plakophilin-related arma-dillo-repeat protein (NPRAP) (Paffenholz and Franke1997). NPRAP appears to be identical to a polypeptideidentified in a yeast two-hybrid screen through itsinteraction with presenilin-1 (Zhou et al., 1997). Of theknown p120ctn/plakophilin-like proteins, only plakophil-ins have been found to accumulate in nuclei, althoughnuclear plakophilins can be extracted during the courseof immunocytochemical staining (see Mertens et al.,1996; Schmidt et al., 1997). It is possible that otherplakophilin-related proteins may accumulate in nuclei.

To test whether the arm repeat region of humanplakophilin-1a (Ppn1) was responsible for its nuclearlocalization, we constructed a series of myc- and greenfluorescent protein (GFP)-tagged forms of the polypep-tide. We find that the nuclear localization of pla-kophilin-1 is due primarily to its N-terminal headdomain, rather than its ‘‘arm-repeats.’’ In the course ofthese studies, we found significant differences betweenthe distribution of myc-reactivity and green fluores-cence in fixed cells. This led us to examine the factorsthat can lead to differences between the apparent and‘‘real’’ distribution of GFP-tagged proteins in culturedcells.

MATERIALS AND METHODSConstruction of Plasmids

A cDNA containing the full coding sequence of humanplakophilin-1a (Ppn1) (Heid et al. 1994) was suppliedby Werner Franke (German Cancer Research Center)and was amplified using polymerase chain reaction(PCR) (5’ primer CCCGAATTCgaaccactcgccgctcaag and3’ primer CCCTCTAGAgaatcgggaggtgaagttc). The am-plified DNA was digested with EcoRI and Xba I (sitesunderlined) and subcloned into the pCS2mt-GFP plas-mid (described in Rubenstein et al., 1997) to formpCS2mt-Ppn1-GFP. The GFP moiety used in our stud-ies contains the S65=T mutation, which enhances GFPfluorescence approximately sixfold (Patterson et al.,1997). In the plasmid, cellular expression is driven by acytomegalovirus promoter; an SP6 promoter can beused to make RNA in vitro (see http://vize222.zo.utexas.edu/Marker_pages/PlasMaps/CS2.html). To create a‘‘head-only’’ construct, the N-terminal head domain(amino acids 1–286) was isolated by PCR (original 5’full-length primer and a 3’ primer CCCTCTAGAcagct-gatagacctgttg), digested with EcoRI and Xba I andsubcloned into the pCS2mt-GFP to form pCS2mt-

Ppn1Head-GFP. A non-GFP-tagged form of the pla-kophilin head domain was generated by digesting thepCS2mt-Ppn1Head-GFP plasmid with Xba I and treat-ing the linearized DNA with the Klenow fragment ofDNA polymerase, which creates an in-frame stop codon.This plasmid is referred to as pCS2mt-Ppn1-Head. A‘‘head-less’’ form (amino acids 287 to 727 of the full-length polypeptide) was amplified (using the 5’ primerCCCGAATTCGggaggcatctgcaagctgg together with thefull-length 3’ primer), digested with EcoRI and Xba Iand subcloned into the pCS2mycGFP plasmid to formpCS2mGFP-Ppn1Arms. The pCS2mGFP and pCS2mt-GFP plasmids are described more fully at http://spot.colorado.edu/,klym/plasmids.html).

Embryo Injection ExperimentsCapped RNA (0.5–1 ng/nl) was made from linearized

pCS2 plasmids using an Ambion (Austin, Texas) mMes-sage mMachine kit following the manufacturer’s instruc-tions. Fertilized egg were generated from hormone-primed females, injected with 10–20 nL of RNA, andmaintained following established laboratory protocols(see Karnovsky and Klymkowsky, 1995). Stages weredefined according to the criteria of Nieuwkoop andFaber (1967). Living embryos were examined by confo-cal microscopy at stages 14–15 (midneurula).

Cell Culture Injection and AnalysisXenopus A6 cells were cultured in 10% fetal calf

serum (Hyclone) in Leibowitz L-15 media (diluted to85% original strength with water) supplemented withMEM non-essential amino acids and antibiotics. Cellswere passaged onto glass-coverslips. Plasmid DNA wasinjected into the nuclei of cell using a simple home-made pressure injection system consisting of a 50-mlglass syringe and a rubber band to supply constantpressure, attached to the end of the needle holder. Forinjection, ‘‘mini-prep’’ plasmid DNA, isolated using theWizard (Promega) system, was processed using a Qia-quick column (Qiagen; PCR protocol); typically 50–100µL of plasmid DNA was resuspended in an equalvolume of 2 X injection buffer (150 mM KCl, 10 mMTris-HCl, 2 mM EDTA, 0.5% sodium azide), yielding afinal DNA concentration of 5–10 µg/ml. At various timesafter injection, the cells were either examined whileliving using a Microimage i308 video camera withimages captured using the ‘‘Minimonitor’’ program anda Apple Power Macintosh 6500/300 computer or werecaptured using a Molecular Dynamics (Sunnyvale, CA)Laser Scanning Confocal microscope.

For immunocytochemistry, cells were fixed with 80%acetone:20% methanol and stained using the anti-mycantibody 9E10 (used diluted 1:10 from spent tissueculture supernatant) (Evan et al., 1985), the pan-cytokeratin antibody C-11 (diluted 1:100) (Sigma, St.Louis, MO), or the anti-vimentin antibody 14h7 (usedneat from spent tissue culture supernatant) (Dent etal., 1989) and Alexa 594-conjugated goat anti-mousesecondary antibodies (diluted 1:200) (Molecular Probes,Inc.). The images presented here were captured using aZeiss (Thornwood, NY) IM35 epifluorescence micro-scope on Ektachrome 400 slide film and digitized usinga Polaroid SprintScan 35 plus scanner or using a Nikonepifluorescence microscope equipped with a Cooke Sen-sicam video camera and the ‘‘SlideBook 2.1’’ program

44 M.W. KLYMKOWSKY

(Intelligent Imaging Innovations, Inc.) using a ApplePower Macintosh 8500/120 computer. These imageswere deconvoluted using the SlideBook program on aApple Power Macintosh G3/266 computer. All imageswere prepared for publication using Adobe Photoshopand Illustrator programs.

RESULTSPlakophilin-1 has an arm repeat/C-terminal domain

of approximately 440 amino acids organized into 10repeats (Hatzfeld et al., 1994; Heid et al., 1994). Basedon the structures of b-catenin (Huber et al., 1997) andkaryopherin-a (Conti et al., 1998) arm repeats areorganized into superhelices of a-helices. Figure 1presents a schematic of plakophilin-1; the arm repeats,as originally defined by Hatzfeld et al. (1994), arepresented as Kyte Doolittle hydrophobicity plots (calcu-lated using the DNA Strider 1.1 program).

To follow the behavior of plakophilin in living cells,we generated myc- and GFP-tagged forms of the protein(Fig. 1). The full coding region of human plakophilin-1cDNA was amplified and subcloned into the pCS2mt-GFP plasmid to generate an NH2-terminal six myc- andCOOH-terminal GFP-tagged form of the protein. mRNAencoding this polypeptide was injected into fertilizedXenopus eggs. In early blastula stage embryos, greenfluorescence was found localized to nuclei (data not

shown). With the development of the embryonic epider-mis (stage 14), green fluorescence was found primarilyin nuclei, but now also associated, albeit weakly, withthe cell-periphery (Fig. 2A). In many nuclei, fluores-cence was distributed uniformly, whereas in others itwas concentrated in discrete intranuclear structures(Fig.. 2A). In contrast to the axis duplication induced bythe injection of plakoglobin-GFP RNA (Rubenstein etal., 1997), injection of mt-Ppn1-GFP RNA produced noobvious effect on early embryonic development (Mer-riam and Klymkowsky, unpublished observations).

To determine which region of the plakophilin-1 poly-peptide was responsible for its nuclear localization, weused PCR to isolate the NH2-terminal ‘‘head’’ andCOOH-terminal ARM-repeat regions (Fig. 1). RNAencoding these polypeptides was injected into fertilizedeggs and the embryos were examined by confocalmicroscopy at stage 14–15 (neurula). The N-terminalhead domain was exclusively nuclear, often formingdiscrete intranuclear inclusions (Fig. 2B,C). In con-trast, the armadillo repeat region was localized to thecytoplasm of embryonic cells and was concentrated atcell-cell boundaries (Fig. 2D,E). It was also readilyapparent that the armadillo domain accumulated innuclei (Fig. 2D–F).

Xenopus A6 cells have a simple epithelial character,express the simple epithelial keratins and vimentin,

Fig. 1. A cartoon of the constructs used in this work. At the top ofthe figure is a schematic representation of the human plakophilin-1asequence with each arm repeat, as determined by Hatzfeld et al.(1994), displayed schematically as a Kyte-Doolittle hydrophobicityplot. Below are the constructs used in this work. They include anN-terminal, six-myc tagged, C-terminal, GFP-tagged version of the

full-length protein (mt-Ppn1-GFP), an N-terminal mycGFP-taggedversion of the C-terminal armadillo-repeat region of the protein(mycGFP-Ppn1ARMS), a N-terminal six-myc tagged, C-terminal GFP-tagged version of the N-terminal head domain (mt-Ppn1HEAD-GFP),and a six-myc tagged version of the N-terminal head domain (mt-Ppn1HEAD).

45GFP-TAGGED PLAKOPHILIN LOCALIZATION

Fig. 2. Plakophilin in Xenopus embryos. RNAs encoding GFP-tagged plakophilins were injected into fertilized eggs. At stage 14(mid-neurula), the surfaces of living embryos were examined by laserscanning confocal microscopy. The mt-PPn1-GFP polypeptide (A)accumulated preferentially in nuclei, often in fluorescent bodies(larger arrow, image is overexposed to visualize membrane-associatedfluorescence). Green fluorescence was also localized to cell-cell bound-aries (A, smaller arrow). Fluorescence from the mtPPn1Head-GFPpolypeptide (B,C) was exclusively nuclear and present in discrete

aggregates (arrows). B and C are distinct confocal sections of the sameembryonic region. Fluorescence from the mycGFP-PPn1ARMS polypep-tide (D–F) was associated with cell-cell boundaries (D, smaller arrow)and could also be seen in nuclei (D, larger arrow). Two confocalsections (E, F) indicate the association of the mycGFP-PPn1ARMSpolypeptide with cell-cell boundaries (E, arrows) and nuclei (F, ar-rows). In embryos not injected with GFP-containing constructs, nofluorescence was visible under these conditions (not shown).

and when confluent make desmosomes with one an-other (see Klymkowsky, 1995). To express the variousforms of plakophilin in these cells, we injected plasmidDNA directly into nuclei (standard DNA transfectionprotocols work only very inefficiently in A6 cells).Within 1 to 2 hours after injection, green fluorescencecould be observed and increased in intensity over thenext 24 hours. All three constructs were injected at afinal DNA concentration of 5–10 µg/ml and all pro-duced comparable levels of green fluorescence.

As in the case of mt-Ppn1-GFP RNA injection intoembryos, green fluorescence from the full-length mt-Ppn1-GFP polypeptide accumulated almost exclusivelyin the nuclei of living A6 cells and typically formed whatappear to be discrete spherical aggregates scatteredthroughout the nuclear volume (Fig. 3A,B). When cellswere fixed and stained with the anti-myc antibody9E10, however, substantial amounts of staining werefound localized to the cytoplasm (Fig. 3C–F). Under theconditions used, staining with the anti-myc antibodyand the Alexa 594-conjugated secondary antibody wascomparable in intensity to green fluorescence, althoughno effort was made to directly compare their relativeintensities. In the nucleus, myc-staining was oftenconfined to the periphery of the nuclear aggregates(arrows in Fig. 3C,D). Presumably the central region ofthese aggregates is inaccessible to the anti-myc anti-body. In addition, we often found regions of myc-staining within the nucleus that did not display greenfluorescence (Fig.. 3E,F). The intensity of myc-stainingin these regions was often similar to that associatedwith the green fluorescent aggregates.

The plakophilin head polypeptide was found largelylocalized to nuclei in living A6 cells (Fig. 4A,B). Itformed irregular aggregates, quite different in theiroverall appearance from the spheroids seen in mt-Ppn1-GFP expressing cells (compare Figs. 4C and3B,C,E). Asin the case of the mt-Ppn1-GFP polypeptide, when fixedcells expressing the mt-Ppn1Head-GFP polypeptidewere stained with the anti-myc antibody 9E10, therewas a substantial amount of cytoplasmic myc-staining(Fig. 4C,D).

Plakophilin-1 has been reported to bind to keratin-type intermediate filaments (Kapprell et al., 1988;Hatzfeld et al., 1994; Smith and Fuchs, 1998). In A6cells, the mt-Ppn1Head-GFP polypeptide was occasion-ally found to decorate cytoplasmic filaments (Fig. 4B).A6 cells contain an extensive and extended keratinfilament network (Fig. 4E). Double staining with amonoclonal anti-keratin antibody revealed that mt-Ppn1Head-GFP fluorescence co-localized with keratinfilaments (Fig. 4A,B,F,G); in many cases the mt-Ppn1Head-GFP decorated keratin network was par-tially collapsed toward the cell center. Removal of theC-terminal GFP-moiety from the mt-Ppn1Head polypep-tide had no noticeable effect on frequency of the interac-tion between the mt-Ppn1Head polypeptide and kera-tin filaments (data not shown).

As in the case of embyronic cells, the ‘‘arm’’ repeatregion of plakophilin was not excluded from A6 cellnuclei (Fig. 5C), but rather accumulated to higherlevels in the cytoplasm (Fig. 5A–C). In particular, themGFP-Ppn1Arms polypeptide was concentrated at thecell cortex, particularly in regions of cell-cell associa-tion and membrane-ruffling (Fig. 5A–C). Moreover, the

distribution of myc-reactivity did not perfectly overlapwith the pattern of green fluorescence in fixed cells (Fig.5 D,E). The effect was less dramatic than that seen withmt-Ppn1-GFP and mt-Ppn1Head-GFP polypeptides,however, possibly due to the fact that mGFP-Ppn1Armshas only a single myc-tag, whereas the mt-PPn1-GFPand mt-Ppn1Head-GFP polypeptides contain a tandemarray of six myc epitopes.

The discordance between green fluorescence andmyc-staining, described here for plakophilin polypep-tides, has also been observed in the case of other doublytagged proteins, particularly the transcription factorsXTcf3, XSlug, XSox17b, human LEF-1, and mouseNeuroD (unpublished data). To test whether this effectwas due at least in part to the time delay between theappearance of the myc-epitope (located at the N-terminus of the nascent polypeptides), the activation ofGFP fluorescence, and the nuclear transport of themature polypeptide, cells were injected with pCS2mt-Ppn1Head-GFP DNA; 18 hours later they were treatedwith 50 µg/ml cycloheximide for 2 hours and then fixedand stained with the anti-myc antibody. Under theseconditions, we found an almost complete disappearanceof cytoplasmic myc-reactivity (Fig. 6). On the otherhand, there were still clear differences in the distribu-tions of myc-reactivity and green fluorescence withinthe nuclei of cycloheximide-treated cells (Fig. 6E,F).

DISCUSSIONWe were originally interested in plakophilin’s nuclear

localization as a way of studying the mechanism in-volved in the nuclear localization of armadillo repeatproteins. Mutational analyses indicate that the ‘‘arm-repeat’’ regions of b-catenin and plakoglobin are suffi-cient to generate both an axis duplication phenotype inXenopus embryos and the polypeptide’s nuclear localiza-tion (Fagotto et al., 1996; Funayama et al., 1995;Karnovsky and Klymkowsky, 1995; Rubenstein et al.,1997). Given the lack of an obvious NLS, it has beenproposed that the nuclear localization of b-catenin andplakoglobin is due to interactions with transcriptionfactors of the TCF family (Behrens et al., 1996; Huberet al., 1996; Molenaar et al., 1996). In turn, the nuclearlocalization of LEF-1 depends upon interactions withkaryopherins through a site distinct from their catenin-binding domain (Prieve et al., 1996, 1998). On the otherhand, b-catenin may localize to nuclei by a karyopherin-independent mechanism (Fagotto et al., 1998).

Like plakoglobin and b-catenin, plakophilins lack anobvious NLS. To determine whether their nuclear local-ization was due to their arm repeat domains, weanalyzed the subcellular localization of four differentmyc- and GFP-tagged forms of plakophilin-1 (Fig. 1).When RNA encoding mt-Ppn-GFP was injected intofertilized eggs, fluorescence from the encoded proteinwas first clearly visible at mid- to late-blastula stages(approximately 10 to 12 hours after injection). At theseearly stages, the protein appeared to be exclusivelynuclear. Endogenous plakophilin-1 has been reportedto be nuclear in the Xenopus oocyte (Schmidt et al.,1997).

The early Xenopus embryo contains a polarly asym-metric keratin filament network but lacks desmosomesuntil after gastrulation (see Gard and Klymkowsky,1998; Perry, 1975; also Dufton and Klymkowsky, unpub-


Fig. 3. Full-length plakophilin in Xenopus A6 cells. Plasmid DNAsencoding GFP-tagged forms of plakophilin were injected into thenuclei of A6 cells; 20 hours later the cells were either photographedunder phase (A) and epifluorescence (B) optics or were fixed (C–F) andstained with the anti-myc antibody. Phase dense aggregates (A, arrow)of fluorescent protein (B) could be seen in the nuclei of cells injectedwith mt-Ppn1-GFP DNA. In fixed cells, we found a substantial amount

of anti-myc fluorescence (D, F) in the cytoplasm, whereas greenfluorescence (C, E) was confined to the nucleus. Typically myc-stainingringed the green fluorescent aggregates, while the centers of theseaggregates were often not stained (C,D, arrows). Even within thenucleus, however, regions of anti-myc staining (F, arrow) and greenfluorescence (E) were often found not to overlap completely.

48 M.W. KLYMKOWSKY

Fig. 4. The N-terminal head domain of plakophilin is localized tonuclei and keratin filaments. Plasmid DNAencoding the mt-Ppn1Head-GFP polypeptide was injected into A6 cells; 20 hours later greenfluorescence was found to localize to nuclei in living cells (A, phase; B,green fluorescence). In a subpopulation of these cells, green fluores-cence was associated with cytoplasmic filaments (B, arrow). In fixedcells injected with mt-Ppn1Head-GFP DNA, green fluorescence wasoften localized to nuclei (C); when such cells were stained with

anti-myc antibody, myc-reactivity was observed in the cytoplasm aswell (D, arrow). A6 cells express keratin filaments, which can bevisualized using an anti-keratin antibody (E). In mt-Ppn1Head-GFPDNA injected cells that display cytoplasmic green fluorescence (F),double staining with an anti-keratin antibody (G) reveals an associa-tion between the plakophilin head polypeptide and the keratin net-work, which often appears to be partially ‘‘collapsed’’ compared to itsnormally extended configuration.

Fig. 5.

lished data). With the formation of the embryonicepidermis, and the appearance of desmosomes, weakgreen fluorescence could also be found at cell-cellboundaries in embryos expressing the mt-Ppn-GFPpolypeptide (Fig. 2A). In contrast, fluorescence from themt-Ppn1Head-GFP polypeptide was exclusively nuclearat all stages (Fig. 2B,C), whereas fluorescence associ-ated with mycGFP-Ppn1Arms was associated preferen-tially with the cell cortex (Fig. 2D–F), although it wasalso clearly able to enter nuclei (Fig. 2F). Whether themycGFP-Ppn1Arms polypeptide is inherently less effi-cient at nuclearly localization, or whether it is activelysequestered in the cytoplasm through interactions withcytoplasmic and membrane proteins, remains unclear.

The behavior of these plakoglobin-derived polypep-tides was similar in Xenopus A6 cells. A6 cells aresimple epithelial in nature and express vimentin andthe simple epithelial keratins K8, K18, and K19 (seeFranz et al., 1983; Gard and Klymkowsky 1998;Klymkowsky, 1995). When expressed in A6 cells, thegreen fluorescence from the mt-Ppn1-GFP polypeptideaccumulated exclusively in nuclei (Fig. 3A). Since pla-kophilin-1 is normally associated with epidermal desmo-somes (Heid et al., 1994), it is not surprising that it wasassociated with cell-cell boundaries in embryonic epider-mis, but not in A6 cells. Within nuclei, the mt-Ppn1-GFP aggregates appeared spherical by light microscopyand were distributed, apparently randomly throughoutthe nuclear volume. In contrast, the mt-Ppn1Head-GFP polypeptide formed more irregular and extendednuclear aggregates in A6 cells (Fig. 4A–D).

One difference between the mt-Ppn1-GFP and mt-Ppn1Head-GFP polypeptides was that the head wasfound to decorate keratin-type intermediate filaments(Fig. 4A,B). This interaction could be restricted to a fewfilaments within the cell or could involve more or lessthe entire keratin filament network, in which case itwas often associated with the ‘‘collapse’’ of the extendedkeratin network (Fig. 4E–G). Previous studies indi-cated that plakophilin-1 binds directly to keratins invitro (Hatzfeld et al., 1994; Kapprell et al., 1988; Smithand Fuchs, 1998). The apparent inability of the full-length protein to interact with keratin filaments raisesthe interesting possibility that the arm repeat domaincan block interactions with keratin filaments and thatbinding of plakophilin to desmosomal cadherins mayregulate plakophilin’s interaction with keratins.

Although the mycGFP-PPn1Arms polypeptide wasnot excluded from A6 cell nuclei (Fig. 5C), the proteinwas found to accumulate in the cytoplasm. Based onblot binding studies, Smith and Fuchs (1998) suggest

that plakophilin-1 binds directly to the desmosomalcadherins desmocollin and desmoglein, as well as to thedesmosomal catenin desmoplakin. In both A6 cells (Fig.5) and embryonic ectoderm cells (Fig. 2D–F), the arm-repeat-containing domain of plakophilin localizes toregions of cell-cell contact and, in A6 cells, to rufflingedges (Fig. 5). However, it is clear that the arm-repeatdomain of plakophilin can also enter nuclei in bothembryonic and A6 cells (Figs. 2D–F, 5). In contrastp120ctn, which interacts with classical cadherins, ap-pears to be exclusively cytoplasmic (Reynolds et al.,1994; A. Reynolds, personal communication). It is pos-sible that p120ctn’s cytoplasmic interactions are strongenough to suppress the weak nuclear translocationassociated with the presence of arm repeats, whereas inplakophilin, the more efficient nuclear localizing activ-ity associated with the N-terminal head domain isenhanced by relatively weak cytoplasmic interactions.

In any case the physiological significance, if any, ofthe nuclear localization of the plakophilins remainsunclear. If they have a role in the control of geneexpression, it does not appear to be associated with thecanonical wnt signaling pathway. Expression of eitherplakophilin-1 (Merriam and Klymkowsky, unpublisheddata) or p120ctn RNA injection (Geis et al., 1998) neithermimics nor suppresses the wnt-like pathway active inthe early Xenopus embryo. Moreover, a human homozy-gous for a severe loss of plakophilin-1 function displaysdefects associated with disruption of desmosomal orga-nization (McGrath et al., 1997) but no obvious defect inthe regulation of gene expression. On the other hand,the presence of a second plakophilin polypeptide (pla-kophilin-2), which is also localized to nuclei (Mertens etal. 1996), raises the possibility of functional redun-dancy in the nuclear functions of these proteins.

An interesting observation to emerge from thesestudies was due to the fact that each of the polypeptidesused had two distinct tags: a GFP-moiety that allowedthe direct visualization of the polypeptide via autofluo-rescence and an myc-epitope, present at theN-terminus, that allowed visualization by indirect im-munocytochemistry. In theory at least, the myc-epitopecan be recognized by the anti-myc antibody as soon as itclears the ribosome. In contrast, the GFP-moiety re-quires a post-translational modification to become fluo-rescent (Heim et al., 1995; Heim and Tsien, 1996). Weconsistently found that the distribution of the myc-reactive epitope differed from the pattern of greenfluorescence, not only for plakophilin-derived polypep-tides (this work) but for a number of other myc- andGFP-tagged polypeptides (data not shown). These dif-ferences are particularly apparent when examiningnuclear proteins, such as mt-Ppn1-GFP (Fig. 3) andmt-PPn1Head-GFP (Fig. 4) but were also apparentwith the cytoplasmic mycGFP-PPn1-Arms polypeptide(Fig. 5).

There are two obvious possibilities to explain thisobservation. The first is that the polypeptides areundergoing proteolysis, such that myc-epitope and theGFP are physically disconnected from one another. Thesecond is that the difference represents the time be-tween the appearance of the myc-epitope and theactivation of the green protein. During this time period,i.e., following the emergence of the nascent polypeptidefrom the ribosome, the polypeptide presumably folds

Fig. 5. The armadillo repeat region of plakophilin localizes thecell-cortex. When DNA encoding the mycGFP-Ppn1ARMS polypeptidewas injected into A6 cells, green fluorescence was found in both thecytoplasm (A,B, two confocal sections of the same cells) and the nuclei(C) of living cells. The nuclear fluorescence was uniform (C) and neverbrighter than the cytoplasmic fluorescence. Cytoplasmic fluorescencewas localized to the cell cortex and was most apparent in regions ofmembrane ruffling. When DNA-injected cells were fixed and stainedwith the anti-myc antibody (E), we observed myc-stained cytoplasmicstructures that were either not fluorescent or only weakly so (D, greenfluorescence). Conversely, there were regions of the cell, particularly atthe ruffling edge, which were fluorescent (D) but not stained stronglyby the anti-myc antibody (E).


Fig. 6. Following the effects of blocking protein synthesis onapparent protein localization. A6 cells were injected with DNA encod-ing the mt-Ppn1Head-GFP polypeptide. After 18 hours, the cells wereeither left untreated (A–D) or were exposed to 50 mg/ml cycloheximide(E,F) for another 2 hours, at which time all cells were fixed and stainedfor the myc epitope. In the control cells green fluorescence (A,C) wasalmost exclusively nuclear, whereas a substantial proportion of the

myc-staining (B,D) was cytoplasmic. In some cells, in fact, myc-staining appeared to be excluded from the nucleus (C,D). After 2 hoursin cycloheximide, cytoplasmic myc-staining (F) had almost completelydisappeared from all cells. The images in B, D, and F were capturedusing identical camera settings and displayed using the same inten-sity scale and so are directly comparable.

52 M.W. KLYMKOWSKY

and, in the case of nuclear protein, is transported intothe nucleus. Ogawa et al. (1995) have reported that acytoplasmic GFP-glucocorticoid receptor fusion proteinrequired 15 minutes, after induction with glucocorti-coid, to become nuclear. In the case of other proteins,the time between the completion of folding and nucleartranslocation is not well defined, at least to our knowl-edge.

To distinguish between these two models, we treatedcells injected with the mt-PPnHead-GFP plasmid withthe translation inhibitor cycloheximide. After 2 hours,we found that cytoplasmic myc-reactivity had largelydisappeared (Fig. 6), arguing that the cytoplasmicstaining observed in our experiments was due to na-scent polypeptides and polypeptides awaiting foldingand transport. Even in the presence of cycloheximide,however, the distribution of myc-staining and greenfluorescence was not always superimposeable (see Fig.6E,F). All of which is to say that the distribution ofgreen fluorescence does not accurately represent thedistribution of all ‘‘tagged’’ polypeptides with the celland may be more accurately considered to represent thedistribution of the mature polypeptide.

ACKNOWLEDGMENTSThe plakophilin RNA embryo injection experiments

were performed by John Merriam and we thank JoshuaMcIlwee and Robert M. Evans for their work on theexpression of mutant plakophilins in mammalian cells.

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Plakophilin, armadillo repeats, and nuclear localization