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Upregulation and Redistribution of E-MAP-115 (Epithelial Microtubule-Associated Protein of 115 kDa) in Terminally Differentiating Keratinocytes is Coincident with the Formation of Intercellular Contacts

Nathalie Fabre-Jonca, Isabelle Viard,* Lars E. French,* and Danie`le Masson Epithelial Network and *Department of Dermatology, University of Geneva, Medical School, Geneva, Switzerland

Microtubules are involved in the positioning and expression significantly increased during the first day, movement of organelles and vesicles and therefore as observed by northern and western blot analysis. play fundamental roles in cell polarization and differen- Parallel immunofluorescence studies showed an early tiation. Their organization and properties are cell-type redistribution of E-MAP-115 from microtubules with specific and are controlled by microtubule-associated a paranuclear localization to cortical microtubules proteins (MAP). E-MAP-115 (epithelial microtubule- organized in spike-like bundles facing intercellular associated protein of 115 kDa) has been identified as a contacts. This phenomenon is transient and can be reversed by Ca2⍣ depletion. Treatment of cells with microtubule-stabilizing protein predominantly expres- 2⍣ sed in epithelial cells. We have used human skin and cytoskeleton-active drugs after raising the Ca con- primary keratinocytes as a model to assess a putative centration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical function of E-MAP-115 in stabilizing and reorganizing localization of these microtubules is dependent on the microtubule network during epithelial cell differen- other microtubules but not on strong interactions with tiation. Immunolabeling of skin sections indicated the actin cytoskeleton or the plasma membrane. The that E-MAP-115 is predominantly expressed in the mechanism whereby E-MAP-115 would redistribute suprabasal layers of the normal epidermis and, in to and stabilize cortical microtubules used for the agreement with this observation, is relatively abundant polarized transport of vesicles towards the plasma in squamous cell carcinomas but barely detectable in membrane, where important reorganizations take basal cell carcinomas. In primary keratinocytes whose place upon stratification, is discussed. Key words: cyto- terminal differentiation was induced by increasing skeleton/microtubules/microtubule-associated proteins/epi- the Ca2⍣ concentration of the medium, E-MAP-115 dermis. J Invest Dermatol 112:216–225, 1999

n interphase cells microtubules are involved in the posi- microtubule cytoskeleton. In isolated cells, microtubules radiate tioning and transport of vesicles and organelles and are from a region near the nucleus, containing the centrioles and the therefore essential for the organization of the intracellular Golgi apparatus, towards the cell periphery. In confluent polarized space. Microtubules are highly dynamic polymers and their cells, the centrioles have split, migrated under the apical plasma cell type-specific stabilization and reorganization are import- membrane (Buendia et al, 1990) and the microtubules no longer Iant for the generation and maintenance of polarity in differentiating originate from the pericentriolar region but are arranged, with their cells such as neurons, myoblasts, and epithelial cells (Kirschner and plus ends towards the basal membrane, in bundles running parallel Mitchison, 1986; Bulinski and Gundersen, 1991; MacRae, 1992; to the apico-basal axis and in sparse basal and dense apical networks Mays et al, 1994; Laferrie`re et al, 1997). Epithelial cell polarization (Bacallao et al, 1989; Gilbert et al, 1991). This reorganization of and differentiation have been mainly studied in the kidney epithelial microtubules is concomitant with their stabilization (Bre´ et al, 1990; cell line MDCK and the colon carcinoma cell line Caco-2 (Bacallao Pepperkok et al, 1990). Although keratinocytes do not display high et al, 1989; Gilbert et al, 1991). When these cells are grown to levels of apico-basal polarity, rearrangement of their microtubule confluency they form specific intercellular junctions that allow network has also been observed upon differentiation in vitro (Lewis demarkation of an apical and a basolateral plasma membrane et al, 1987; Girolomoni et al, 1992). domain. In parallel, there is a fundamental rearrangement of the Microtubule dynamics and organization are presumably con- trolled by microtubule-associated proteins (MAP) whose expression levels vary with the cell type and differentiation status. MAP were Manuscript received June 16, 1998; revised October 15, 1998; accepted initially identified in neuroneal tissues, because the brain is an for publication November 2, 1998. important source of microtubules, based on their property to Reprint requests to: Dr. Danie`le Masson, Laboratoire Re´seau Epithelium, copurify with microtubules during cycles of polymerization and Center Me´dical Universitaire, 1 rue Michel-Servet, CH-1211 Geneva 4. depolymerization. These are MAP1, MAP2, and tau (Matus, 1988; Abbreviations: E-MAP-115, epithelial microtubule-associated protein of Wiche, 1989; Maccioni and Cambiazo, 1995; Mandelkow and 115 kDa; MAP, microtubule-associated protein. Mandelkow, 1995). Few non-neuroneal MAP have been character-

0022-202X/99/$10.50 · Copyright © 1999 by The Society for Investigative Dermatology, Inc. 216 VOL. 112, NO. 2 FEBRUARY 1999 E-MAP-115 IN TERMINALLY DIFFERENTIATING KERATINOCYTES 217 ized so far. MAP4, the best characterized of these MAP, is a family 1.2 U dispase per ml. The epiderm was then separated from the derm and of proteins translated from alternatively spliced RNA that display treated with 0.1% trypsin in phosphate-buffered saline for 20 min at 37°C. different tissue distributions (Chapin et al, 1995). MAP have at first Trypsin activity was then inhibited by adding one volume of Dulbecco’s been shown to modulate the dynamic behavior of microtubules modified Eagle’s medium containing 10% fetal calf serum and the cell suspension was filtered, homogeneized, and rinsed twice with the same in vitro. Transfection of MAP cDNA into fibroblasts and insect medium. Primary cells were plated at a density of 7 ϫ 104 per cm2 in cells has been widely used to demonstrate the role of MAP in the FAD culture medium (25% Ham’s F12 medium and 75% Dulbecco’s control of microtubule dynamics in cells and for the characterization modified Eagle’s medium supplemented with 10% fetal calf serum, 0.13 mM of these molecules (Lee, 1993; Hirokawa, 1994). adenine, 0.142 U insulin per ml, 4 nM triiodothyronine, 2 mM hydrocortis- E-MAP-115 is a MAP predominantly expressed in epithelial cell one, 292 mg glutamine per ml, 10 ng epidermal growth factor per ml, lines (Masson and Kreis, 1993) and in the epithelial component of 0.84 mg cholera toxin per ml, 100 U penicillin per ml, 100 mg streptomycin several organs (Fabre-Jonca et al, 1998). A role for E-MAP-115 in per ml, 0.25 mg fungizone per ml) on mitomycin growth-arrested human stabilizing and reorganizing the microtubule cytoskeleton during fibroblasts as feeder layers. The medium was changed every 2–3 d until epithelial cell polarization and differentiation has been proposed keratinocytes reached about 80%–90% confluency. Monolayers were then either subcultured in defined keratinocyte-SFM medium (GibcoBRL, based on the observations that: (i) E-MAP-115 expression increases Basel, Switzerland) containing 0.09 mM Ca2ϩ, or resuspended in freezing with cell polarization in epithelial cell lines and developing organs; medium (10% dimethyl sulfoxide in fetal calf serum) and stored in liquid (ii) transient transfection of fibroblasts that do not have detectable nitrogen. Cells were routinely grown to 80%–90% confluency at 37°C in levels of E-MAP-115 with the cDNA encoding this protein induces an atmosphere of 5% CO2 in SFM and the medium was changed every stabilization and reorganization of their microtubules; (iii) the 2–3 d. Cells used for experiments were always under the third passage and interaction of E-MAP-115 with microtubules decreases at the onset were plated at a density of 12.5 ϫ 102 per cm2 in 100 mm Petri dishes or of mitosis when microtubules become more dynamic (Masson and 24 wells plates containing coverslips that had previously been treated with Kreis, 1995). coating medium (0.01 mg fibronectin per ml, 0.03 mg collagen per ml, Human epidermis is a stratified epithelium in which basal 0.1 mg bovine serum albumin per ml in SFM). Terminal differentiation was induced by adding CaCl2 to a final concentration of 1.5 mM. proliferative keratinocytes progressively stop dividing and enter Differentiating cells were further grown for up to 10 d. terminal differentiation as they progress through the suprabasal layers to finally produce a protective outer coat of dead terminally Immunocytochemistry MoAb D9C1 against E-MAP-115 was used differentiated cells (Fuchs, 1990; Fuchs and Byrne, 1994). Epidermal for immunocytochemistry on paraffin sections of Bouin’s fixed human terminal differentiation can be partially reproduced in vitro using epidermis sections using a streptavidin and biotin complex (streptABCom- human primary keratinocytes. Keratinocytes grown in low [Ca2ϩ] plex/HRP; Dako, Glostrup, Denmark) according to the manufacturer’s medium are prevented from stratifying but the onset of terminal instructions and 3-amino-9-ethylcarbazole as a chromogen for peroxydase. differentiation still occurs in about 30%–50% of the cells that display an increased size, express involucrin, and are finally expelled from Gel electrophoresis and immunoblotting Keratinocytes were tryp- the basal layer. Raising the Ca2ϩ concentration in the culture sinized, counted, and then lyzed in hot sample buffer and samples medium triggers terminal differentiation of most keratinocytes; they corresponding to equivalent numbers of cells were analyzed on 8% stop proliferating, develop cellular junctions, form multilayers, and polyacrylamide gels according to Laemmli (1970), followed by transfer of proteins to nitrocellulose as previously described (Masson and Kreis, 1993). express other terminal gene products such as keratins K1, K10, or Immunoblots were developed using an ECL-chemiluminescent detection filaggrin (Fuchs and Green, 1980; Watt and Green, 1982; Boyce kit (Amersham, Arlington Heights, IL). and Ham, 1983; Hennings and Holbrook, 1983; Watt, 1984; Pillai et al, 1990). We have used human skin and primary keratinocytes Total RNA extraction and northern blot analysis Total RNA was to assess a possible function of E-MAP-115 in stabilizing and extracted from keratinocytes according to the procedure of Chomczynski reorganizing the microtubule cytoskeleton during epithelial cell and Sacchi (1987), denatured in the presence of glyoxal and dimethyl differentiation. Our results indicate that E-MAP-115 expression sulfoxide, electrophoresed through a 1% agarose gel in 10 mM phosphate correlates with the degree of terminal differentiation of keratinocytes buffer pH 6.8, and transferred to Hybond-N nylon membrane (Amersham) in vivo and in vitro. Interestingly, in primary keratinocytes E-MAP- by capillarity. Prehybridation and hybridation were performed at 65°C in 115 is typically relocalized to stable cortical microtubule bundles 50% formamide, 50 mM Pipes, 0.8 M NaCl, 2 mM ethylenediamine tetraacetic acid, 2.5 ϫ Denhardt’s, 0.1 mg denatured salmon sperm DNA facing intercellular contacts early during terminal differentiation. per ml and 0.1% sodium dodecyl sulfate. cRNA probes were synthesized Taken together our results strongly support a role for E-MAP-115 using α32P-UTP (Amersham) from Bluescript KS plasmids containing in setting up the basis of the architecture of terminally differentiating either a cDNA fragment coding for a truncated form of human E-MAP- keratinocytes. 115 (Mb, human E-MAP-115 lacking amino acids 81–426; Masson and Kreis, 1993) linearized at NheI or the cDNA coding for the 18S RNA. MATERIALS AND METHODS Hybridized RNA were visualized by autoradiography. Materials Commercially available monoclonal antibodies directed against actin (MAB1501, Chemicon, Temecula, CA), desmoplakin I and II (DP1 Immunofluorescence Keratinocytes grown on glass coverslips were & 2–2.15, Boehringer, Mannheim, Germany), filaggrin (BT-516, BTI, fixed either in methanol for 5 min at –20°C or in 3% paraformaldehyde Stoughton, MA), and involucrin (I9018, Sigma, St Louis, MO) were used and processed as described (Masson and Kreis, 1995). In some experiments, for immunoblotting and immunofluorescence labeling. Rabbit polyclonal cells were fixed in methanol after preextraction in 0.5% Triton X-100, antibodies were used to detect tyrosinated tubulin (anti-T13) and detyrosin- 80 mM K-Pipes pH 6.8, 5 mM ethyleneglycol-bis(β-aminoethyl ether)- ated tubulin (anti-T12) (Kreis, 1987) and human E-MAP-115 was detected N,N,NЈ,NЈ-tetraacetic acid (EGTA), 1 mM MgCl2. Epifluorescence micro- by different polyclonal (R2) or monoclonal (D9C1, D6B2) antibodies scopy was performed using a Zeiss 63 ϫ 1.4 planapo oil immersion (Masson and Kreis, 1993). Horseradish peroxydase-coupled goat anti-mouse objective on a Zeiss axiophot microscope (Carl Zeiss, Oberkochen, or anti-rabbit IgG (BioRad Labs, Mu¨nchen, Germany) and rhodamine- or Germany). Digitized fluorescence images were obtained using a CoolView fluorescein-conjugated anti-mouse or anti-rabbit F(abЈ)2 fragment IgG camera (Photonic Science) coupled to the microscope. In some experiments (Jackson Immunoresearch, West Grove, PA) were used as secondary cells were analyzed using a confocal laser scanning microscope, Zeiss LSM antibodies for immunoblotting and for immunofluorescence labeling, 410 invert, equipped with Ar (488 nm) and HeNe (543 nm) lasers. respectively. F-actin was labeled using rhodamine-coupled phalloidin. Nocodazole, taxol, and cytochalasin D stock solutions at 5 mM, 10 mM, RESULTS or 1 mM, respectively, were prepared in dimethyl sulfoxide and stored E-MAP-115 distribution in normal human skin and in epi- at –20°C. dermal carcinomas Immunocytochemical analysis of sections Cell culture Epidermal keratinocytes were isolated from human breast of normal human skin for E-MAP-115 revealed the presence of or abdominal skin. Briefly, skin was cut into 0.25 cm2 pieces that were the protein in the epidermis, whereas it was absent from cells of incubated overnight at 4°C in Dulbecco’s modified Eagle’s medium the dermis (Fig 1a). Interestingly, higher levels of E-MAP-115 supplemented with 10% fetal calf serum, 20 mM HEPES pH 7.3, and were detected in the terminally differentiating suprabasal than in 218 FABRE-JONCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

1988). Taken together, these observations indicate a correlation between E-MAP-115 expression and the degree of epidermal differentiation in vivo. E-MAP-115 expression is upregulated upon terminal differ- entiation of human primary keratinocytes Terminal differen- tiation of human primary keratinocytes was induced by raising the Ca2ϩ concentration in the culture medium and monitored by immunoblotting using involucrin and profilaggrin, as early and late differentiation markers, respectively, for 10 d (Fig 2a). As observed by others (Rice and Green, 1979; Banks-Schlegel and Green, 1981; Watt and Green, 1981; Watt, 1984), involucrin, which is one of the major components of the cornified envelope, could already be detected in keratinocytes grown in low [Ca2ϩ] medium; however, it was µ2.5 times more abundant after1dinhigh [Ca2ϩ] medium. Profilaggrin, the precursor of the intermediate filament-aggregating protein filaggrin synthesized in cells of the granular layer (Fleckman et al, 1985; Steven et al, 1990), was detected from 3 d of growth in high [Ca2ϩ] medium onwards and levels of this protein continuously increased with time. E-MAP-115 expression was followed in parallel western (Fig 2a) and northern (Fig 2b) blot analysis and the level of the protein and corresponding mRNA quantitated by densitometric scanning (Fig 2c). Interestingly, E- MAP-115 protein was already detected in proliferating keratinocytes but its cellular content was increased 3-fold after1dofculture in high [Ca2ϩ] medium and 4-fold after 3 d. From this time point onwards, E-MAP-115 expression did not change significantly. A protein with slightly lower apparent mass (about 110 kDa) was also recognized by anti-E-MAP-115 antibodies. A similar band appears upon polarization and differentiation of intestinal Caco-2 cells and has been shown to correspond to an isotype of E-MAP-115 (Fabre- Jonca and Masson, manuscript in preparation). E-MAP-115 mRNA levels also increased during culture in high [Ca2ϩ] medium but to a lesser extent. In contrast to E-MAP-115 and the differentiation marker proteins, actin and tubulin, which were also detected by immunoblotting, appeared to remain at constant expression levels (Fig 2a). Thus, this analysis clearly demonstrates a quantitative upregulation of E-MAP-115 expression upon keratinocyte terminal differentiation. Induction of keratinocyte terminal differentiation triggers the intracellular redistribution of E-MAP-115 The intracellu- lar distribution of E-MAP-115 and of microtubules in proliferating and terminally differentiating keratinocytes was analyzed by double- immunofluorescence microscopy. In keratinocytes grown in low [Ca2ϩ] medium, microtubules formed a dense network surrounding the nucleus and were weakly and discontinuously stained for E- MAP-115 (Fig 3a, b). Raising the Ca2ϩ concentration in the culture medium triggered the formation of intercellular contacts and, interestingly, microtubules reorganized into bundles directed towards some of these contacts (Fig 3d, f). Concomitantly, E- MAP-115 was completely relocalized to these cortical spike-like Figure 1. Localization of E-MAP-115 in normal human skin and microtubule bundles (Fig 3c, e). E-MAP-115 redistribution was an in carcinomas. E-MAP-115 was detected in sections of normal human early process because it was observed in some cells already 1 h after skin (a), basal cell carcinoma (b), and squamous cell carcinoma (c)by raising the calcium concentration (Fig 3c, d) and in all cells after immunocytochemistry using MoAb D9C1 as described in Materials and 6h(Fig 3e, f), when no increase in the levels of this protein Methods. Note the predominant expression of E-MAP-115 in the upper could be detected (as monitored by immunoblotting; not shown). layers of the normal skin, its absence from most cells of a basal cell carcinoma, and its preferential distribution in the center of the squamous Interestingly, as observed in sections of immunolabeled cells cell carcinoma tumor mass. Scale bar: 150 µm. obtained by confocal microscopy (Fig 4), E-MAP-115-positive microtubule bundles were always associated with domains of the cell cortex overlapping neighboring cells (i.e., in the upper confocal sections of stratifying cells). From 3 d in high [Ca2ϩ] onwards, when the proliferative basal layers of keratinocytes. Analysis of sections stratification had occurred, typical spike-like cortical microtubule of basal cell carcinomas and squamous cell carcinomas were done bundles could not be observed anymore and microtubules had in parallel. The hyperproliferative basal-like keratinocytes of basal reorganized into a dense network that was homogeneously labeled cell carcinoma were negative for E-MAP-115, contrary to cells of for E-MAP-115 (results not shown). Taken together these results the suprabasal layers in which E-MAP-115 was still present (Fig 1b). hint at a role of E-MAP-115 in the process of stratification. Most of the spreading squamous-like keratinocytes of squamous cell carcinoma were positive for E-MAP-115 (Fig 1c). Strikingly, Intracellular redistribution of E-MAP-115 and upregulation highest levels of the protein were found in cells of the center of of its expression coincide with an increase in microtubule the tumor that are known to be more differentiated (Stoler et al, stability Increased expression of E-MAP-115 upon cell differenti- VOL. 112, NO. 2 FEBRUARY 1999 E-MAP-115 IN TERMINALLY DIFFERENTIATING KERATINOCYTES 219

assess a possible correlation between E-MAP-115 expression and changes in microtubule dynamics in terminally differentiating keratinocytes, cells grown for different times in high [Ca2ϩ] medium were treated with the microtubule-depolymerizing drug nocodazole and double-labeled for E-MAP-115 and tubulin. Some very short microtubules positive for E-MAP-115 could still be observed in cells maintained in low [Ca2ϩ] medium (Fig 5a, b); however, strikingly, after 6 h in high [Ca2ϩ] medium, nocodazole-resistant microtubules were both more numerous and longer (Fig 5c, d). These stable microtubules were always positive for E-MAP-115 but, surprisingly, they were not organized as cortical bundles (as observed in nontreated cells, see Fig 3c, e) but as individual microtubules randomly distributed in the cytoplasm. This observa- tion suggested either that E-MAP-115-positive microtubules were stabilized and their cortical localization was sensitive to nocodazole- treatment, or that E-MAP-115-positive cortical microtubule bundles had depolymerized and E-MAP-115 had then redistributed to other remaining stable microtubules. Further increase in microtu- bule stability was observed from 24 h in high [Ca2ϩ] medium onwards (not shown). These experiments show an increase in microtubule stability coinciding with an upregulation of E-MAP- 115 expression; however, microtubule stability already increased after 6 h in high [Ca2ϩ] medium, when E-MAP-115 levels were not detectably increased. The intracellular redistribution of E- MAP-115 shortly after raising the Ca2ϩ concentration of the medium may involve changes in the properties of this protein. E- MAP-115 microtubule-binding activity is modulated by phospho- rylation during the cell cycle (Masson and Kreis, 1995); whether some post-translational modifications of this protein at the onset of terminal differentiation finally result in changes in microtubule dynamics remains to be investigated. Stabilization of microtubules was further assessed by analyzing keratinocytes for the presence of detyrosinated α-tubulin (glu- tubulin, a marker of stabilized microtubules). In cells maintained in low [Ca2ϩ] or grown for 6 h and 24 h in high [Ca2ϩ] medium, microtubules appeared negative for glu-tubulin (not shown); how- ever, glu-tubulin labeling does not strictly correlate with microtub- ule stabilization. As observed by others in MDCK cells (Bre´ et al, 1987), appearance of glu-tubulin on stabilized microtubules is not immediate. Moreover, tubulin may be further digested by a carboxypeptidase, thus removing the epitope recognized by antiglu- tubulin antibodies (e.g., in ∆2 tubulin; Paturle-Lafaneche`re et al, 1994). To investigate the absence of staining for glu-tubulin in keratinocytes, cells grown in high [Ca2ϩ] medium for different periods of time were treated with the microtubule-stabilizing drug taxol for 1 h and processed for immunolabeling with anti-E-MAP- 115 and anti-glu-tubulin antibodies. In cells grown for6hinhigh [Ca2ϩ] medium (Fig 5e, f), E-MAP-115-positive microtubules were still organized in cortical bundles but these appeared to have a broader distribution at the cell periphery and to be shorter than in cells not treated with the drug (see Fig 3e, f). Interestingly, these Figure 2. Upregulation of E-MAP-115 protein and mRNA levels E-MAP-115-positive microtubule bundles were always positive for in terminally differentiating keratinocytes. Monolayers of human glu-tubulin that was itself concentrated on these bundles (Fig 5e, keratinocytes grown in low [Ca2ϩ] medium were further cultured in high 2ϩ f). Hence, taxol treatment of keratinocytes shortly after raising the [Ca ] medium for 1–10 d. Levels of different proteins were monitored 2ϩ by immunoblotting (a) and of E-MAP-115 mRNA by northern blot Ca concentration in the medium revealed a higher stability analysis (b). (a) Total proteins extracted from differentiating keratinocytes of these microtubules as compared with E-MAP-115-negative at days 0, 1, 3, and 10 (lanes 1, 2, 3, 4, respectively) were analyzed by microtubules. In this context, bundling of microtubules has been immunoblotting using antibodies against involucrin, profilaggrin, E-MAP- observed to coincide with their stabilization (Lee and Brandt, 1992). 115, actin, and tubulin (MoAb I9018, MoAb BT-516, MoAb D6B2, Nonetheless, stabilization of E-MAP-115-positive microtubules in MAB1501, and anti-T13, respectively). (b) Northern blot analysis of total keratinocytes is not sufficient to result in their modification by the cellular RNA isolated from differentiating keratinocytes at days 0, 1, 3, detyrosinating carboxypeptidase. and 10 (lanes 1, 2, 3, 4, respectively) using E-MAP-115 or human 18S cRNA probes as described in Materials and Methods.(c) Quantitation of E- Interactions of E-MAP-115-positive microtubules An MAP-115 mRNA (gray bars) and protein (black bars) levels by important consequence of raising the Ca2ϩ concentration in the densitometric scanning of the autoradiograms shown in (b) and (a), respectively. culture medium is to allow the establishment of intercellular contacts and junctions (Geiger et al, 1983; Hennings and Holbrook, 1983; Watt et al, 1984; Schmidt et al, 1994). A possible colocalization ation has been observed in the intestinal cell line Caco-2 and a of vinculin, an actin-binding protein found in intercellular adherens role for this protein in stabilizing the microtubules of differentiating junctions of keratinocytes (Green et al, 1987), with E-MAP-115 epithelial cells has been proposed (Masson and Kreis, 1993). To was investigated; however, no clear colocalization between the two 220 FABRE-JONCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. E-MAP-115 is relocalized to the cell periphery of keratinocytes exposed to high [Ca2⍣] medium. Human keratinocytes grown in low [Ca2ϩ] (a, b) or in high [Ca2ϩ] medium for 1 h (c, d)or6h(e,f) were fixed in methanol and double immunofluorescence labeling was performed with MoAb D9C1 against E-MAP-115 (a, c, e) and rabbit anti-tubulin antibodies (b, d, f). Scale bar:3µm.

proteins could be established (not shown). Then, the formation of nocodazole for 3 h and analyzing them by immunofluorescence desmosomes in cells undergoing terminal differentiation was fol- microscopy. As shown in Fig 6(e, f), although nocodazole treatment lowed by immunolabeling for desmoplakin I and II. In proliferating prevented the redistribution of E-MAP-115-positive microtubules keratinocytes, desmoplakin was diffusely distributed throughout to the cell periphery, it did not inhibit desmoplakin relocalization the cytoplasm (not shown). Two hours after raising the Ca2ϩ at the membrane upon calcium stimulation. This observation is in concentration, when E-MAP-115 had started to redistribute to the agreement with results obtained by others, ruling out a fundamental cell periphery, few desmoplakin-positive dots were observed near role of microtubules in the formation of desmosomes in MDCK adjacent cells (not shown). Interestingly, after 6 h, when E-MAP- cells (Pasdar et al, 1992). 115 was exclusively found on cortical microtubule bundles (Fig 6a), Because integrins and nondesmosomal cadherins are linked to desmoplakin patches appeared to align along these microtubules as the actin cytoskeleton and these interactions are required for well as along other microtubules that were negative for E-MAP- stratification (Braga et al, 1995), a possible role of microfilaments 115 (Fig 6b; double-labeling for desmoplakin and tubulin not in the cortical localization of E-MAP-115-positive microtubules shown). A transient interaction between desmosomes and micro- was analyzed by treating keratinocytes with cytochalasin D. When tubules has been shown by others in MDCK cells (Wacker et al, the drug was added before the [Ca2ϩ] elevation and maintained 1992). To investigate a possible relationship between E-MAP-115 throughout the experiment, E-MAP-115-positive microtubules and desmoplakin and the reversibility of their redistribution, cells were no longer relocalized to the cell periphery (result not shown); grown in high [Ca2ϩ] medium for 6 h were exposed to EGTA for however, their cortical localization was not prevented when the different periods of time and processed for immunolabeling. After actin cytoskeleton was disassembled a few hours after raising 1 h of treatment, cortical E-MAP-115-positive microtubule bundles the [Ca2ϩ] in the culture medium (Fig 6g, h). Hence, intact had started to disassemble into individual microtubules randomly microfilaments are required to initiate E-MAP-115-positive micro- distributed in the cytoplasm, whereas desmoplakin was still found tubules redistribution, probably due to their role in the establishment as patches aligned along microtubules (not shown). After2hof of intercellular junctions (Green et al, 1987; O’Keefe et al, 1987; EGTA treatment all E-MAP-115 was found back on individual Braga et al, 1995), but they are not necessary to maintain this perinuclear microtubules (Fig 6c) and desmoplakin had started to localization, ruling out a strong interaction between E-MAP-115 be internalized (Fig 6d). Taken together these observations indicate and the actin cytoskeleton. Interestingly, when cytochalasin D was that E-MAP-115 redistribution to the apical cortex of keratinocytes applied to keratinocytes grown in high [Ca2ϩ] medium, E-MAP- is concomitant with the formation of intercellular contacts and is 115-positive microtubules formed very long and thin centripetal an earlier and more dynamic process than desmoplakin redistribu- protrusions, presumably resulting from a decreased mechanical tion. A possible role of E-MAP-115-positive microtubules in the resistance at the plasma membrane, suggesting that these microtub- formation of desmosomes in early differentiating keratinocytes was ules are either growing at their plus end or transported towards further assessed by culturing cells in high [Ca2ϩ] medium containing the cell periphery along other cytoskeletal components. This VOL. 112, NO. 2 FEBRUARY 1999 E-MAP-115 IN TERMINALLY DIFFERENTIATING KERATINOCYTES 221

Figure 4. E-MAP-115-positive micro- tubule bundles are restricted to the upper domain of terminally dif- ferentiating keratinocytes. Confocal sectioning images of human keratinocytes grown for 6 h in high [Ca2ϩ] medium, fixed in methanol, and stained for E-MAP- 115 (red) and tubulin (green) as described in Fig 3 are shown. Optical sectioning was adjusted 1 µm(a), 2 µm(b), 3 µm(c), or 4 µm(d) above the plane of adhesion. Scale bar:3µm.

observation together with the effect of nocodazole on the localiz- and primary keratinocytes to assess a putative function of E-MAP- ation of E-MAP-115-positive microtubule bundles hinted at a 115 in epithelial cell differentiation. We report that E-MAP-115 transport of these polymers along more labile microtubules via expression is upregulated during epidermal and primary keratinocyte microtubule-dependent motors. E-MAP-115-positive microtub- terminal differentiation and that the protein present in proliferating ules, however, could also be transported along stabilized microtub- keratinocytes is very rapidly relocalized to stable microtubule ules because, as described above, E-MAP-115-positive microtubules bundles facing intercellular contacts at the onset of terminal in taxol-treated keratinocytes formed even more abundant but differentiation. Taken together, our results support a role for E- shorter bundles. A model whereby E-MAP-115-positive micro- MAP-115 in setting up the basis of the architecture of terminally tubules would be transiently redistributed to the cell periphery differentiating keratinocytes. along other microtubules will be discussed below. In normal skin E-MAP-115 is predominantly expressed in the Taken together, these observations indicate that the cortical suprabasal layers of the epidermis that consist of cells that are no distribution of E-MAP-115-positive microtubules in keratinocytes longer proliferating and have entered terminal differentiation. shortly after raising the Ca2ϩ concentration is concomitant with Moreover, E-MAP-115 is barely detectable in hyperproliferative the formation of intercellular contacts and depends on other keratinocytes of basal cell carcinoma that are blocked in an microtubules, but not on strong interactions with the actin cytoskel- undifferentiated state, and shows a perturbed distribution in hyper- eton or the plasma membrane, and suggest that E-MAP-115- proliferative cells of squamous cell carcinoma that display an positive cortical microtubules, although they do not appear to be aberrant balance between growth and differentiation. In normal strictly necessary for the formation of desmosomes, could be skin, keratins K5 and K14 are synthesized in the basal layer and involved in the targeting of material required to build the apical their expression is downregulated as the cells differentiate in domain of stratifying keratinocytes. Further experiments are contrary to K1 and K10, which are only found in the suprabasal required to identify molecules or structures interacting with E- layers of the epidermis (Fuchs and Green, 1980; Fuchs, 1995). MAP-115-positive microtubules. Interestingly, the expression of epidermal keratins has been shown to vary in different epidermal diseases correlating with the differenti- DISCUSSION ation status of cells. K1 was shown to be barely detectable in or Changes in the organization of microtubules accompanying epithe- absent from most tumor cells of basal cell carcinoma, whereas K14 lial cell differentiation have been well studied in MDCK or Caco- was present in all cells (Stoler et al, 1988). Squamous cell carcinoma 2 cells (Bacallao et al, 1989; Gilbert et al, 1991), and similar tumors were observed to be homogenously positive for K14, rearrangements have been observed in primary keratinocytes (Lewis whereas K1 was found predominantly in cells of the center that et al, 1987; Zamansky et al, 1991). E-MAP-115 is the only are more differentiated (Weiss et al, 1984; Stoler et al, 1988). protein described so far that could control the stabilization and E-MAP-115 displays an expression pattern similar to keratin K1 in reorganization of microtubules in epithelial cells (Masson and Kreis, these diseases and could therefore be used as an additional marker 1993; Fabre-Jonca et al, 1998). Here, we have used human skin to assess the differentiation status of cells. 222 FABRE-JONCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 5. Effect of microtubule-active drugs on E-MAP-115 intracellular distribution and microtubule stability. Human keratinocytes maintained in low [Ca2ϩ](a,b) or grown in high [Ca2ϩ](c– f) medium for 6 h were treated for 1 h with 10 µM nocodazole (a–d)or10µM taxol (e, f) and then fixed in methanol. Nocodazole-treated cells were preextracted with 0.5% Triton buffer as described in Materials and Methods before fixation. Double immunofluorescence labeling was performed using MoAb D9C1 against E- MAP-115 (a, c, e) and rabbit anti- tyrosinated tubulin antibodies T13 (b, d)or rabbit anti-detyrosinated tubulin antibodies T12 (f). Scale bar:3µm.

Primary keratinocytes can be induced to stratify and complete Hence, our observations hint at a role of E-MAP-115 in the terminal differentiation by raising the Ca2ϩ concentration of the process of stratification. culture medium. We have taken advantage of this system to Formation of both adherens junctions and desmosomes is rapidly follow E-MAP-115 expression and intracellular localization during induced in primary keratinocytes upon raising the Ca2ϩ concentra- terminal differentiation. Like others we have observed a complete tion and stratification ensues. Adherens junctions can be detected reorganization of the microtubule network (Girolomoni et al, before desmosomes and desmosome formation as well as stratifica- 1992); within 1 h after raising the Ca2ϩ concentration, a subclass of tion depend on functional nondesmosomal cadherins (Hennings microtubules is rearranged into cortical bundles facing intercellular and Holbrook, 1983; Watt et al, 1984; Green et al, 1987; O’Keefe contacts. Interestingly, E-MAP-115, which is localized to randomly et al, 1987; Wheelock and Jensen, 1992; Lewis et al, 1994; Zhu organized perinuclear microtubules in proliferating cells, is found and Watt, 1996). These intercellular junctions are connected to exclusively on these peripheral microtubule bundles at an early stage different components of the cytoskeleton. The cytoplasmic plaque of in vitro terminal differentiation. Furthermore, these microtubule of adherens junctions is linked to microfilaments and the actin bundles are stabilized and microtubule stabilization increases with cytoskeleton is required for the redistribution of nondesmosomal further cellular differentiation. As observed in sections of immunola- cadherins to cell–cell contacts and for keratinocyte stratification beled cells obtained by confocal microscopy, E-MAP-115-positive following calcium stimulation (O’Keefe et al, 1987; Braga et al, microtubule bundles are always associated with domains of the cell 1995). Apart from being linked to intermediate filaments, desmo- cortex overlapping neighboring cells. This polarized localization of somes may transiently interact with microtubules as suggested from E-MAP-115-positive bundles appears to be transient because they observations done in MDCK cells in which CLIP-170, a protein are no longer observed in more differentiated cells. A role of linking endosomes to microtubules (Rickard and Kreis, 1990; microtubules in initiating the formation of focal contacts and Pierre et al, 1992), has been proposed to mediate this interaction facilitating the establishment of long-lived contacts during fibroblast (Wacker et al, 1992). Thus, we investigated the relationship of E- locomotion has been demonstrated (Rinnerthaler et al, 1988). MAP-115-positive microtubules with adherens junctions, desmo- Interestingly, E-MAP-115 was excluded from microtubules facing somes, and the actin cytoskeleton. Immunofluorescence labeling the edge of a wounded keratinocyte monolayer (data not shown), using vinculin as a marker of adherens junctions revealed only indicating that its intracellular localization is indeed related to the occasional colocalization of E-MAP-115-positive bundles with this formation of new intercellular contacts and not to cell migration. protein. Interestingly, the forming desmosomal plaques located at VOL. 112, NO. 2 FEBRUARY 1999 E-MAP-115 IN TERMINALLY DIFFERENTIATING KERATINOCYTES 223

Figure 6. Relationship between E- MAP-115-positive microtubule bundles and desmoplakin or the actin cytoskeleton. Keratinocytes grown in high [Ca2ϩ] medium for 6 h were immediately fixed in cold methanol (a, b) or treated for 2 h with 4 mM EGTA before fixation (c, d), and double immunofluorescence labeling was performed using rabbit anti-E-MAP- 115 antibodies (a, c) and MoAb DP1 & 2– 2.15 against desmoplakin (b, d). Keratinocytes grown for3hinhigh [Ca2ϩ] medium supplemented with 10 µM nocodazole were fixed in cold methanol and processed for double immunofluorescence labeling using rabbit anti-E-MAP-115 antibodies (e) and MoAb DP1 & 2–2.15 (f). Keratinocytes grown in high [Ca2ϩ] medium for 6 h were treated for 1 h with 4 µM cytochalasin D and fixed in paraformaldehyde (g, h). E-MAP-115 and actin were visualized using MoAb D9C1 (g) and rhodamine-coupled phalloidin (h), respectively. Scale bar:3µm.

the apical cortex of stratifying keratinocytes were most often aligned The properties of E-MAP-115-positive microtubules and the with E-MAP-115-positive microtubule bundles. Nonetheless, this mechanism of redistribution of these microtubules to the cell cortex colocalization was transient; the redistribution of E-MAP-115- were analyzed by immunofluorescence labeling of cells treated with positive microtubules to the cell cortex was an earlier and more cytoskeleton active drugs. These experiments indicated that E- dynamic process than desmoplakin relocalization, ruling out a role of MAP-115-positive microtubule bundles are stabilized, that their desmosomes in E-MAP-115-positive microtubules reorganization. cortical localization depends on other microtubules but not on Reciprocally, E-MAP-115-positive microtubules did not appear to strong interactions with the plasma membrane and the actin be necessary for desmosome formation. Our experiments indicated cytoskeleton and, finally, that the redistribution of E-MAP-115- that the initial redistribution of E-MAP-115-positive microtubules positive microtubules to the cell periphery is a dynamic process. to the cell cortex was dependent on intact microfilaments; however, Transport of microtubules along preexisting polymers has been the fact that the actin cytoskeleton was not required to maintain demonstrated in axonal growth (Ahmad and Baas, 1995; Miller this cortical localization after the formation of junctions suggests and Joshi, 1996), and molecular motors of the superfamily of that this component of the cytoskeleton is only indirectly involved kinesins may be involved in this process (Nakagawa et al, 1997; in E-MAP-115 redistribution (i.e., the actin cytoskeleton is neces- Yang et al, 1997; Hirokawa, 1998). Reorganization of microtubules sary for the formation of junctions) and excludes a strong interaction in differentiating epithelial cells may involve a similar mechanism. between this protein and microfilaments. Therefore, the redistribu- Based on our observations we propose the following model for the tion of E-MAP-115 to cortical microtubule bundles is most redistribution of E-MAP-115 upon raising the [Ca2ϩ] concentration probably related to the establishment of novel intercellular contacts of the culture medium: in stratifying keratinocytes and is more coincident with the forma- (a) In cells grown in low [Ca2ϩ] medium, low levels of E-MAP- tion of adherens junctions than of desmosomes, but the requirement 115 are sparsely and homogenously distributed on randomly of adherens junctions for this process remains to be established. organized microtubules that explore the whole cytoplasmic 224 FABRE-JONCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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