localization of phosphorylated erm proteins

INTRODUCTION

The ERM protein family consists of three closely relatedproteins, ezrin, radixin and moesin (Bretscher, 1983; Pakkanenet al., 1987; Tsukita et al., 1989; Lankes and Furthmayr, 1991;Sato et al., 1992). They are thought to function as generalcross-linkers between plasma membranes and actin filaments(Arpin et al., 1994; Tsukita et al., 1997a,b; Bretscher et al.,1997). In cultured epithelial and fibroblastic cells, they areexpressed and localized at specialized regions where actinfilaments are densely associated with plasma membranes, suchas cleavage furrows, microvilli, ruffling membranes, and cell-cell/cell-matrix adhesion sites, whereas in tissues the levels andcombination of their expression vary considerably (Bretscher,1983; Pakkanen et al., 1987; Lankes et al., 1988; Tsukita et al.,1989; Sato et al., 1991, 1992; Berryman et al., 1993; Takeuchiet al., 1994; Henry et al., 1995). The suppression of ERMexpression with antisense oligonucleotides affectedmicrovillus formation as well as cell-cell/cell-matrix adhesion(Takeuchi et al., 1994), and the introduction of a dominant-negative construct of ezrin and radixin impaired cortical actinorganization and cytokinesis, respectively (Henry et al., 1995;Martin et al., 1995).

ERM proteins bind to actin filaments mainly at their COOH-termini (Algrain et al., 1993; Edwards et al., 1994; Turunen etal., 1994; Henry et al., 1995; Martin et al., 1995; Pestonjamaspet al., 1995), and to integral membrane proteins such as CD44,CD43, ICAM-2 and ICAM-3 at their NH2-terminal half(Tsukita et al., 1994; Hirao et al., 1996; Serrador et al., 1997;Yonemura et al., 1998). Several lines of evidence haveindicated that in the cytoplasm NH2- and COOH-terminalhalves of ERM proteins are associated intra- and/orintermolecularly, which suppresses their binding ability tointegral membrane proteins and actin filaments, respectively(Gary and Bretscher, 1995; Henry et al., 1995; Magendantz etal., 1995; Martin et al., 1995; Hirao et al., 1996; Tsukita et al.,1997a,b; Bretscher et al., 1995; Berryman et al., 1995). Suchinactivated ERM proteins in the cytoplasm are thought to beactivated by some signals to function as actin filament/plasmamembrane cross-linkers just beneath the plasma membranes.

ERM proteins were reported to be phosphorylated attyrosine as well as serine/threonine residues under variousphysiological conditions (Bretscher, 1989; Kreig and Hunter,1992; Urushidani et al., 1989; Chen et al., 1995; Kondo et al.,1997; Nakamura et al., 1995; Matsui et al., 1998). Recent invitro biochemical studies revealed that the phosphorylation of

1149Journal of Cell Science 112, 1149-1158 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0256

Ezrin/radixin/moesin (ERM) proteins are thought to playan important role in organizing cortical actin-basedcytoskeletons through cross-linkage of actin filaments withintegral membrane proteins. Recent in vitro biochemicalstudies have revealed that ERM proteins phosphorylatedon their COOH-terminal threonine residue (CPERMs) areactive in their cross-linking activity, but this has not yetbeen evaluated in vivo. To immunofluorescently visualizeCPERMs in cultured cells as well as tissues using a mAbspecific for CPERMs, we developed a new fixation protocolusing trichloroacetic acid (TCA) as a fixative.Immunoblotting analyses in combination withimmunofluorescence microscopy showed that TCAeffectively inactivated soluble phosphatases, whichmaintained the phosphorylation level of CPERMs during

sample processing for immunofluorescence staining.Immunofluorescence microscopy with TCA-fixed samplesrevealed that CPERMs were exclusively associated withplasma membranes in a variety of cells and tissues, whereastotal ERM proteins were distributed in both the cytoplasmand plasma membranes. Furthermore, the amounts ofCPERMs were shown to be regulated in a cell and tissuetype-dependent manner. These findings favored the notionthat phosphorylation of the COOH-terminal threonineplays a key role in the regulation of the cross-linkingactivity of ERM proteins in vivo.

Key words: Ezrin, Radixin, Moesin, ERM, Phosphorylation,Trichloroacetic acid

SUMMARY

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with

their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative

Ken Hayashi 1, Shigenobu Yonemura 1,*, Takeshi Matsui 1, Sachiko Tsukita 1,2 and Shoichiro Tsukita 1

1Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan2College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto 606, Japan*Author for correspondence (e-mail: [email protected])

Accepted 10 February; published on WWW 23 March 1999

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their COOH-terminal threonine residue (T567 in ezrin, T564in radixin and T558 in moesin) was important for regulationof their cross-linking activity. When platelets were activated bythrombin, T558 in moesin was specifically phosphorylated(Nakamura et al., 1995). T564 in radixin was phosphorylatedby Rho-associated kinase (Rho-kinase) in vitro. T567 in ezrin,T564 in radixin and T558 in moesin were phosphorylated inSwiss 3T3 cells in a Rho-dependent manner, and in T564-phosphorylated radixin the interaction between its NH2- andCOOH-terminal halves was suppressed to stabilize its activeform (Matsui et al., 1998). These findings suggested that ERMproteins with their COOH-terminal threonine residuephosphorylated (CPERMs; COOH-terminally phosphorylatedERM proteins) represent the active form of ERM proteins.Although phosphorylated ERM proteins were biochemicallyshown to be translocated from the cytoplasm to apical cellprotrusions in NIH-3T3 cells (Shaw et al., 1998) and thelocalization of CPERMs was discussed in certain cases(Nakamura et al., 1996; Oshiro et al., 1998), our knowledgeregarding the precise localization of CPERMs in vivo is stilllimited.

In this study, we examined the subcellular distribution ofCPERMs by immunofluorescence microscopy. Previously, weraised a mAb, 297S, that specifically recognized CPERMs butnot non-phosphorylated ERM proteins on immunoblotting(Matsui et al., 1998). However, by immunofluorescencemicroscopy this mAb did not give any signals in cultured cellsfixed with formaldehyde or organic solvents such as methanol,ethanol or acetone. We presumed that dephosphorylation orchemical modification by fixation prevented theimmunoreaction, and thus we searched for a novel fixationprotocol to inactivate enzymes such as phosphatases withoutstrong chemical modification. Trichloroacetic acid (TCA) wasfound to be an appropriate fixative for this purpose. In thisstudy, we evaluated this newly developed TCA fixationprotocol biochemically as well as morphologically, and thenexamined the subcellular distributions of CPERMs not only incultured cells but also in tissues.

MATERIALS AND METHODS

CellsMouse epithelial MTD-1A cells (Enami et al., 1984; Hirano et al.,1987), mouse fibroblastic L cells (Earl, 1943), mouse NIH3T3fibroblasts clone 5611 (Jainchill et al., 1969), dog epithelial MDCKcells (Gaush et al., 1966), rat 3Y1 fibroblasts (Kimura et al., 1975),rat basophilic leukemia RBL-2H3 cells and human epidermoidcarcinoma A431 cells (Fabricant et al., 1977) were cultured in DMEsupplemented with 10% FCS. MTD-1A, L, and MDCK cells weregifts from Dr M. Takeichi (Kyoto University, Kyoto, Japan), andNIH3T3 clone 5611, RBL-2H3 and A431 cells were obtained fromthe Japanese Cancer Research Resources Bank (Tokyo, Japan).

Antibodies and other molecular markersThe following anti-ERM antibodies were used; mouse mAb (CR22)with higher affinity for moesin than for ezrin and radixin (Sato et al.,1991), rabbit pAb (TK89) which recognizes COOH-termini of allERM proteins (Kondo at al., 1997), and rat mAb (297S) whichspecifically detects the phosphorylated threonine at the COOHterminus of each ERM protein (T567 of ezrin, T564 of radixin andT558 of moesin) (Matsui et al., 1998). We also used mouse anti-tubulin mAb (DM1A) (Sigma Chemical Co., St Louis, MO), mouse

anti-rat ZO-1 mAb (T8-754) (Itoh et al., 1991), mouse anti-phosphotyrosine mAb (clone 4G10, Upstate Biotechnology. Inc.,Lake Placid. NY), mouse anti-actin mAb (clone C4, BoehringerMannheim), rhodamine-conjugated WGA (wheatgerm lectin) (E-YLaboratories, Inc., San Metro, CA), and 4′6-diamidino-2-phenylindole dehydrochloride (DAPI).

Fixation protocolsTCA: trichloroacetic acid (TCA) was dissolved with distilled water tomake 100% (w/v) stock solution and stored at 4°C, which was dilutedto 10% with distilled water before use. Cells cultured on coverslipsor small tissue blocks from DDY mice were immersed in ice-cold 10%TCA for 15 minutes for cells or 1 hour for tissues, then washed withPBS containing 30 mM glycine (G-PBS) three times.

Formaldehyde: fixation was performed with 1% or 4%paraformaldehyde in 0.1 M Hepes buffer (pH 7.4) at roomtemperature for 15 minutes for cells or 1 hour for tissues, followed bywashing with G-PBS.

Ethanol/acetone: 95% ethanol was used for fixation at −20°C for30 minutes for cells or 1 hour for tissues, then the samples wereexposed to acetone at room temperature for 1 minute for cells or 5minutes for tissues, followed by washing with G-PBS.

Methanol: methanol was used at −20°C for 15 minutes for cells or1 hour for tissues, followed by washing with G-PBS.

Immunofluorescence microscopyAfter TCA fixation and washing, tissue blocks were mounted inTissue-Tek O.C.T. Compound (Sakura Finetechnical Co., Ltd, Tokyo)and were frozen in liquid nitrogen. Frozen samples were cut intosections 5 µm thick on a cryostat, and put on coverslips coated withpoly-L-lysine, then air-dried. These TCA-fixed sections and TCA- orformaldehyde-fixed cultured cells were treated with 0.2% Triton X-100 in G-PBS for 15 minutes, and washed with G-PBS three times.These samples (and also cultured cells fixed with organic solvents)were soaked in blocking solution (G-PBS containing 10% FCS and1% bovine serum albumin) for 1 hour, and incubated with primaryantibodies for 1 hour. They were washed three times with G-PBS, thenincubated with secondary antibodies for 30 minutes. In someexperiments, rhodamine-conjugated WGA or DAPI was added to thesecondary antibody solution. Cy2-labeled donkey anti-mouse IgGantibody, Cy2-labeled goat anti-rabbit IgG antibody, and Cy3-labeledgoat anti-rat IgG antibody (Amersham) were used as secondaryantibodies. Samples were washed three times, mounted in 90%glycerol-PBS containing 0.1% para-phenylendiamine and 1% n-propylgalate, and observed using a Zeiss Axiophot photomicroscope(Carl Zeiss, Oberkochen, Germany). Images were recorded with acooled CCD camera (SenSys 0400, 768×512 pixels; Photometrics,Tucson, AZ) controlled by a Power Macintosh 7600/132 and thesoftware package IPLab Spectrum V3.1 (Signal Analytics Corp.,Vienna, VA). An MRC 1024 confocal fluorescence microscope (Bio-Rad Laboratories) equipped with a Zeiss Axiophot IIphotomicroscope was also used.

Post-fixation immunoblottingCells or tissues were fixed, permeabilized and washed according tothe protocols for immunofluorescence described above. They werethen dissolved in sample buffer for SDS-PAGE and sonicated using aSONIFER 250 (Branson), followed by boiling for 3 minutes. Sampleswere separated by SDS-PAGE in 10% polyacrylamide gels. Afterelectrophoresis, proteins were transferred onto nitrocellulosemembranes and incubated with first antibodies. Bound antibodieswere then visualized using biotinylated secondary antibody followedby avidin-conjugated alkaline phosphatase.

Dephosphorylation experimentsMTD-1A cells cultured in 10 cm dishes were fixed with methanol.After methanol was completely removed, cells were incubated with 4

K. Hayashi and others

1151Localization of phosphorylated ERM proteins

ml G-PBS for 10 minutes. This G-PBS extract was recovered andstocked. Cells cultured on coverslips or 35 mm dish were fixed withTCA, permeabilized and washed three times. These cells wereincubated with the G-PBS-extract at 37°C for 1 hour, and processedfor immunofluorescence microscopy or immunoblotting.

RESULTS

Development and evaluation of a fixation protocolwith TCATo examine the subcellular distribution of COOH-terminallyphosphorylated ERM proteins (CPERMs), we doubly stainedMTD-1A cells with rat mAb 297S and mouse mAb CR22; theformer specifically recognizes CPERMs on immunoblotting(Matsui et al., 1998) and the latter recognizes moesin inimmunofluorescence microscopy. Cultured MTD-1A cells

were previously shown to express all ERM proteins that werecolocalized (Takeuchi et al., 1994). Unexpectedly, when cellswere fixed with formaldehyde or organic solvents such asmethanol, ethanol or acetone, 297S did not give any significantsignals, whereas CR22 stained mainly apical microvilli (Fig.1A). We then examined how to preserve the antigenicity for297S during fixation, and finally found that fixation with 10%TCA was appropriate for this purpose (Fig. 1Aa,b). In TCA-fixed cells, the appearance of CR22-positive apical microvilliwas the same as that in cells fixed with formaldehyde ororganic solvents, and most of these microvilli were clearlystained with 297S. These findings indicated that TCA is a goodfixative in terms of preservation not only of structures at thelight microscopic level but also of antigenicity for 297S forimmunofluorescence microscopy.

To further evaluate TCA as a fixative, we analyzed samplesfixed and processed for immunofluorescence microscopy, by

Fig. 1.Evaluation of TCA-fixation. (A) MTD-1A cells were fixed with 10%TCA (a and b), 4% formaldehyde (c and d), 1% formaldehyde (e and f),methanol (g and h), or ethanol/acetone (i and j), then doubly stained with anti-ERM mAb, CR22 (a,c,e,g,i) and anti-CPERM mAb 297S (b,d,f,h,j). Only inTCA-fixed cells was the anitgenecity for 297S preserved. Bar, 20 µm. (B) Post-fixation immunoblotting analysis. MTD-1A cells were fixed with 10% TCA(TCA), 1% formaldehyde (1%FA), 4% formaldehyde (4%FA), ethanol/acetone(EtOH/acetone), or methanol (MtOH), washed with PBS and permeabilized,followed by SDS-PAGE and immunoblotting with rabbit anti-ERM pAb(TK89) or 297S. Unfixed cell lysate (control) and methanol-fixed cells withoutG-PBS wash (MtOH (unwashed)) were also examined. The phosphorylationlevel of ERM proteins in unfixed cells was maintained only in those fixed withTCA. When methanol-fixed cells were not washed with G-PBS, thephosphorylation level was partially maintained.

A B

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immunoblotting. We called this analysis post-fixationimmunoblotting. As shown in Fig. 1B, equal amount ofproteins from MTD-1A cells, which were fixed with eachfixative, permeabilized and washed for immunofluorescencemicroscopy, were applied to SDS-PAGE, followed byimmunoblotting with TK89 (specific for all ERM proteins) or297S. TK89 recognized ezrin, radixin and moesin irrespectiveof the fixation protocol, although in formaldehyde-fixed cellsmany positively stained high molecular weight bands wereobserved probably due to formaldehyde-induced aggregationof ERM proteins. In contrast, 297S recognized three bands ofERM proteins (CPERMs) only in unfixed as well as TCA-fixedcell lysates.

When cells were fixed with methanol followed by G-PBS-washing, they gave no signals on 297S immunoblotting, butinterestingly if methanol-fixed cells were not washed with G-PBS prior to SDS-PAGE clear 297S-positive ERM bands weredetected (Fig. 1B, lane MtOH (unwashed)). Thus, wepresumed that CPERMs might be dephosphorylated during G-PBS washing by some endogenous phosphatases that were notinactivated by methanol fixation. To evaluate this hypothesis,the following experiments were performed (Fig. 2): MTD-1Acells were fixed with methanol and incubated with G-PBS. ThisG-PBS-extract, which we expected to contain putativephosphatases, was applied to TCA-fixed and permeabilizedcells, followed by incubation for 1 hour at 37°C. When thesecells were immunolabeled with 297S, no signals were detectedon immunofluorescence microscopy (Fig. 2Ad) as well asimmunoblotting (Fig. 2B). Then, we concluded that methanolcannot inactivate endogenous phosphatases, whereas TCAdoes. This may explain why TCA preserved the antigenicity ofCPERMs for 297S.

This TCA fixation can be used generally forimmunocytochemistry due to its good preservation of

structures. In TCA-fixed cultured MTD-1A cells, phasecontrast microscopy showed no obvious structural damagesand relatively good preservation of organelles and cellularfibers (Fig. 3A). Furthermore, many markers such as anti-tubulin mAb (DM1A) (Fig. 3B), an anti-ZO-1 mAb (T8-754)(Fig. 3C) and rhodamine-conjugated WGA (Fig. 3D) stainedstructures in TCA-fixed cells. Phalloidin did not bind to actinfilaments, but, instead, anti-actin mAb (C4) was available tovisualize actin filaments in TCA-fixed cells (data not shown).Mouse anti-phosphotyrosine mAb (4G10) stained intenselycellular structures such as focal contacts in TCA-fixed cells aswell as in formaldehyde-fixed cells but only weakly inmethanol-fixed cells (data not shown). Post-fixationimmunoblotting with 4G10 revealed that TCA-fixed,permeabilized and washed cells contained similar amount ofphosphotyrosine to unfixed cells and that the amount ofphosphotyrosine in methanol-fixed cells decreased duringPBS-washing (data not shown).

Subcellular distribution of CPERMs in cultured cellsIn all TCA-fixed cultured cells, CPERMs were easilyvisualized by 297S. We then performed confocal microscopicanalyses of 297S/CR22-doubly-stained MTD-1A, 3Y1fibroblasts, and RBL-2H3 leukemia cells (Fig. 4). In thesecells, CR22 intensely stained their surface microvilli, cell-to-cell boundaries and cleavage furrows in addition to the faintbut significant staining of the cytoplasm (Fig. 4A-C). Incontrast, 297S specifically stained microvilli, cell-to-cellboundaries and cleavage furrows, but did not give any signalsin the cytoplasm (Fig. 4D-F). In composite images, the biasedlocalization of CPERMs to plasma membranes was clearlyvisualized (Fig. 4G-I). These findings indicated that whenERM proteins were phosphorylated at their COOH-terminalthreonine residue, they were preferentially targeted to the


Fig. 2.Phosphatase activity in methanol-fixed cells. (A) MTD-1A cells were fixed with methanol then extracted with G-PBS. 10% TCA-fixedMTD-1A cells were incubated with this G-PBS extract (c and d) or control G-PBS (a and b) for 1 hour at 37°C, followed by doubleimmunofluorescence staining with anti-ERM mAb, CR22 (a and c) and 297S (b and d). Bar, 20 µm. (B) TK89 (lane 1 and 2) or 297S (lane 3and 4) immunoblotting of MTD-1A cells incubated with the G-PBS extract (lane 2 and 4) or the control G-PBS (lane 1 and 3).

A B


cytoplasmic surfaces of plasma membranes, probably tofunction as cross-linkers between actin filaments and plasmamembranes.

Distribution of CPERMs in tissuesThe effect of TCA fixation on the phosphorylation level ofCPERMs was examined in tissues by post-fixationimmunoblotting (Fig. 5). After ethanol/acetone fixation, aconventional fixation protocol for frozen sections, CPERMswere not detected by 297S on immunoblotting in either theliver or kidney. In contrast, in TCA-fixed liver or kidney,phosphorylated radixin or ezrin was detected, respectively,probably because ezrin or radixin predominated among ERMproteins in these tissues. Thus, also in tissues, the TCA fixationprotocol appeared to be useful for examination of subcellulardistribution of CPERMs.

Frozen sections of various TCA-fixed tissues were thendoubly stained with TK89 pAb and 297S mAb. In most tissues,297S-positive CPERMs were colocalized with TK89-positivetotal ERM proteins, although the former appeared to be morehighly concentrated at membrane structures such as microvillithan the latter (Fig. 6). In intestinal epithelial cells as well askidney proximal tubules, CPERMs were exclusivelyconcentrated at brush border microvilli (Fig. 6A-D). CPERMswere also abundantly detected in glomeruli in the kidney (Fig.6C,D) and in mesothelial cells (data not shown). In endothelialcells, moesin was reported to predominate among ERMproteins (Berryman et al., 1993; Schwartz-Albeiz et al., 1995),and 297S also stained endothelial cells very intensely invarious tissues such as the kidney (Fig. 6C,D), cerebrum (Fig.6E,F) and tongue (Fig. 7A,B). In peripheral nerves, TK89 aswell as 297S strongly stained microvilli of Schwann cells atthe node of Ranvier (Fig. 6G,H).

In some types of cells, a clear discrepancy between TK89-and 297S-staining patterns was observed. Blood capillaries inintestinal villi (Fig. 6A,B), plasma membranes of smooth

muscle cells (data not shown), stratified epithelium (Fig.7A,B), plasma membranes of granulosa cells and thecytoplasm of oocytes (Fig. 7C,D) were stained with TK89 butnot with 297S, showing that CPERMs were also localized justbeneath the plasma membranes in tissues. These resultssuggested that phosphorylation of the COOH-terminalthreonine residue of ERM proteins is elaborately regulated ina cell-type specific manner.

Decrease of the amount of CPERMs during anoxiaIt was reported that the anoxic injury on renal proximaltubules caused dephosphorylation at serine or threonineresidues of ezrin with concomitant microvillar breakdown(Chen et al., 1995). Therefore, we applied the TCA fixationprotocol with 279S mAb to examine this phenomenon, sinceit would give a very good evaluation for the TCA fixation. Toobtain anoxic conditions, small blocks of kidney or smallintestine were incubated in PBS for 1 hour at 37°C. Then,these samples were fixed with TCA and processed forimmunostaining or immunoblotting with 297S mAb or TK89pAb (Fig. 8). In the kidney proximal tubules, either TK89pAb- or 297S mAb staining mostly disappeared, suggestingthe dephosphorylation of ERM proteins with concomitantbreakdown of microvilli under the anoxic conditions (Fig.8Aa,b). This dephosphorylation was also confirmed byimmunoblotting (Fig. 8B). Interestingly, ERM proteins inendothelial cells of blood capillaries were not markedlydephosphorylated. In the small intestine, TK89 staining at thebrush border did not change significantly under anoxicconditions, suggesting that microvillar breakdown was notinduced. However, 279S-staining revealed that ERM proteinsin the brush border were almost completely dephosphorylated(Fig. 8Ac,d). This dephosphorylation was again confirmed byimmunoblotting (Fig. 8B). These findings confirmed thevalidity of the TCA-fixation protocol in tissues as well as thebehavior (dephosphorylation) of ERM proteins during anoxic

Fig. 3.TCA-fixed MTD-1Acells. (A) Phase-contrastimage; (B)immunofluorescence stainingwith anti-tubulin mAb, DM1A;(C) immunofluorescencestaining with anti-ZO-1 mAb,T8-754; (D) fuorescencestaining with rhodamine-conjugated WGA (Golgiapparatus marker). Bar, 20 µm.

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injury which was demonstrated previously in a biochemicalstudy.

DISCUSSION

We previously raised a mAb (297S) that specificallyrecognized ERM proteins with the COOH-terminal threonineresidue phosphorylated (CPERMs) on immunoblotting(Matsui et al., 1998). However, when we examined thesubcellular distribution of CPERMs by immunofluorescencemicroscopy using this mAb, we faced a technical difficulty inthat 297S mAb gave no signals in cells/tissues which were

fixed either with formaldehyde or organic solvents. In thisstudy, we first attempted to develop a fixation protocol for 297Simmunostaining, and found that TCA was very potent as afixative for this purpose.

TCA has been widely used as a protein coagulant inbiochemistry and also as a minor fixative for histology.However, there have been few studies using TCA as a fixativefor immunostaining; Leong et al. (1979) compared severalfixatives, including 5% TCA, on immunostaining, but found noadvantages of TCA fixation. As shown in this study, TCAinactivates phosphatases very effectively as compared to otherfixatives, which may be why TCA fixation was good for 297Sstaining. We confirmed that tyrosine phosphatase was


Fig. 4.Confocal micrographs of MTD-1A (A,D,G), 3Y1 (B,E,H) and RBL-2H3 (C,F,I) cells. Cells were fixed with 10% TCA and doublystained with anti-ERM mAb, CR22 (green; A, B and C) and 297S (red; D,E and F). G, H and I, composite images. Cytoplasm (large arrows)showed only the CR22 signal, whereas microvilli (small arrows) were stained by both CR22 and 297S. Bar, 20 µm.


Fig. 5.Post-fixation immunoblotting analysis of kidney andliver with TK89 and 297S. Mouse kidney and liver were fixedwith 10% TCA (kidney-TCA or liver-TCA) orethanol/acetone (kidney-EtOH/acetone or liver-EtOH/acetone), permeabilized and washed with G-PBS,followed by SDS-PAGE and immunoblotting.

Fig. 6.TK89 and 297S doubleimmunofluorescence stainingof frozen sections of mousesmall intestine (A and B),kidney cortex (C and D),cerebrum (E and F) and sciaticnerve (G and H). Arrows in Cand D, brush borders of renalproximal tubules; arrowheadsin C and D, endothelial cells;asterisks in C and D,glomerulus; arrows in G and H,nodes of Ranvier. Bar, 20 µm.

1156 K. Hayashi and others

Fig. 7.TK89 and 297S doubleimmunofluorescence stainingof frozen sections of mousetongue (A and B) and ovary (Cand D). Arrows in A and B,endothelial cells in dermis;arrowheads in A and B,stratified squamous epitheliumof epidermis; asterisks in C andD, oocytes. Bar, 20 µm.

Fig. 8.Decrease in the amounts ofCPERMs during anoxia. (A) Small blocksof kidney (a and b) or small intestine (cand d) were incubated in PBS at 37°C for1 hour, then frozen sections of TCA-fixedtissue blocks were doubly stained withTK89 (a and c) and 297S (b and d).Arrows in a and b, brush borders ofproximal tubules; arrowheads in a and b,endothelial cells; arrows in c and d, brushborders of intestinal epithelial cells. Bar,20 µm. (B) Small blocks of kidney orsmall intestine were incubated in PBS at37°C for 1 hour, then processed for SDS-PAGE and immunoblotting (PBSincubation +) or directly processed forSDS-PAGE and immunoblotting (PBSincubation -) with TK89 or 297S.

A

B


inactivated by TCA fixation, although this type of phosphatasewas also affected by formaldehyde fixation.

Considering that TCA preserved the structural integrity ofcells at least at the light microscopic level, and that TCA didnot affect the staining of other antibodies and reagents (exceptfor rhodamine-phalloidin), TCA fixation should be regarded asuseful for immunostaining, not only with phosphorylatedprotein-specific antibodies, but also for that with antibodieswhich did not immunostain samples fixed by conventionalmethods.

The TCA fixation protocol was evaluated in this study byanalyzing cultured cells on immunoblots that were fixed,permeabilized and washed according to the protocols forimmunofluorescence microscopy. In the pioneering work byHerman et al. (1981), to search for a fixation protocolappropriate to stain cells with anti-actin or anti-myosinantibodies, protein compositions of fixed cells were carefullycompared in CBB-stained SDS-polyacrylamide gels as well ason immunoblots. Clark and Damjanov (1986) examined theeffect of fixatives on immunoblotting for keratin polypeptides.There were also several studies in which proteins wereextracted and examined from fixed and mounted tissues (Ikedaet al., 1998). However, what we referred to in the present study‘post-fixation immunoblotting’ has not been used widely forevaluation of immunostaining of cells. As shown in this study,post-fixation immunoblotting provides important informationfor the evaluation of fixation protocol as well as the imagesobtained in immunofluorescence microscopy.

TCA fixation allowed us to examine the subcellulardistribution of CPERMs in cells/tissues in detail. CPERMswere detected in all cell lines examined throughout the cellcycle. In most types of cultured cells and tissues, CPERMswere mostly localized just beneath the plasma membranes,whereas considerable amounts of total ERM proteins weredistributed not only at plasma membranes but also in thecytoplasm. Of course, in some cases, subcellular distributionsof CPERMs were not distinguishable from those of total ERMproteins. To date, biochemical analyses have revealed thatERM proteins function as general cross-linkers betweenplasma membranes and actin filaments (Arpin et al., 1994;Tsukita et al., 1997a,b; Bretscher et al., 1997) and suggestedthat CPERMs represent active forms of ERM proteins in termsof their cross-linking activity (Matsui et al., 1998).Furthermore, serine/threonine phosphorylation of ERMproteins in lysophospatidic acid-treated (i.e. Rho-activated)NIH3T3 cells (Shaw et al., 1998) or their dephosphorylationin anoxic kidney epithelial cells (Chen et al., 1995)/in Fas-induced apoptotic cells (Kondo et al., 1997) werebiochemically shown to increase or decrease, respectively, thedegree of association of ERM proteins with plasmamembranes. Therefore, our present observations with 297SmAb in TCA-fixed cells/tissues, i.e. the biased concentrationof CPERMs at plasma membranes, supported these previousbiochemical findings. Conversely, the present immunostainingdata demonstrated conclusively that the phosphorylation of theCOOH-terminal threonine is one of the key regulatorymechanisms of the cross-linking activity of ERM proteins invivo.

Some types of cells such as capillaries in intestinal villi,smooth muscle cells, epidermal cells of stratified epitheliumand granulosa cells surrounding oocytes expressed significant

amounts of ERM proteins, but in these cells CPERMs were notdetected. These observations indicated that thephosphorylation level of the COOH-terminal threonine residueof ERM proteins is elaborately regulated in a cell type-specificmanner. Rho-kinase (ROCK; Leung et al., 1995; Matsui et al.,1996; Ishizaki et al., 1996) was suggested to be one of thekinases responsible for phosphorylation of the COOH-terminalthreonine residue of ERM proteins in vitro as well as in vivo(Matsui et al., 1998). Recently, protein kinase C-θ was alsoreported to specifically phosphorylate the COOH-terminalthreonine residue of ERM proteins (Pietromonaco et al., 1998).Since Rho-kinase is regulated under the control of Rho, theRho-dependent signaling pathway may determine the amountsof CPERMs within cells. On the other hand, our results andthose of a previous study by Chen and Mandel (1997) revealedfairly strong phosphatase activity for CPERMs in thecytoplasm. It is therefore likely that the phosphorylation levelof CPERMs is also dependent on the activities of these putativephosphatases. Identification and characterization of thesephosphatases is an important issue for future studies.

It is still unclear whether the COOH-terminalphosphorylation of ERM proteins is the cause or the result ofactivation of ERM proteins. Rho-kinase could notphosphorylate an inactive full-length radixin efficiently at leastin vitro. However, the threonine-phosphorylation of theCOOH-terminal half of radixin affected its direct binding tothe NH2-terminal half of radixin, suggesting that thephosphorylation maintains the active state of ERM proteins bysuppressing their head-to-tail association (Matsui et al., 1998).The novel TCA fixation protocol presented in this study willbe helpful in understanding the ERM-activation mechanism indetail within cells.

We thank all the members of our laboratory (Department of CellBiology, Faculty of Medicine, Kyoto University) for helpfuldiscussions. This study was supported in part by a Grant-in-Aid forCancer Research and a Grant-in-Aid for Scientific Research (A) fromthe Ministry of Education, Science and Culture of Japan.

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localization of phosphorylated erm proteins

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