FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2021287789> ?p ?o ?g. }

• W2021287789 endingPage "225" @default.
• W2021287789 startingPage "216" @default.
• W2021287789 abstract "Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed. Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed. epithelial microtubule-associated protein of 115 kDa microtubule-associated protein In interphase cells microtubules are involved in the positioning and transport of vesicles and organelles and are therefore essential for the organization of the intracellular space. Microtubules are highly dynamic polymers and their cell type-specific stabilization and reorganization are important for the generation and maintenance of polarity in differentiating cells such as neurons, myoblasts, and epithelial cells (Kirschner and Mitchison, 1986Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (922) Google Scholar;Bulinski and Gundersen, 1991Bulinski J.C. Gundersen G.G. Stabilization and posttranslational modification of microtubules during cellular morphogenesis.Bioessays. 1991; 13: 285-293Crossref PubMed Scopus (236) Google Scholar;MacRae, 1992MacRae T.H. Towards an understanding of microtubul function and cell organization: an overview.Biochem Cell Biol. 1992; 70: 835-841Crossref PubMed Scopus (58) Google Scholar;Mays et al., 1994Mays R.W. Beck K.A. Nelson W.J. Organization and function of the cytoskeleton in polarized epithelial cells: a component of the protein sorting machinery.Curr Opin Cell Biol. 1994; 6: 16-24Crossref PubMed Scopus (135) Google Scholar;Laferrière et al., 1997Laferrière N.B. MacRae T.H. Brown D.L. Tubulin synthesis and assembly in differentiating neurons.Biochem Cell Biol. 1997; 75: 103-117Crossref PubMed Scopus (69) Google Scholar). Epithelial cell polarization and differentiation have been mainly studied in the kidney epithelial cell line MDCK and the colon carcinoma cell line Caco-2 (Bacallao et al., 1989Bacallao R. Antony C. Dotti C. Karsenti E. Stelzer E.H.K. Simons K. The subcellular organization of Madin-Darby Canine Kidney cells during the formation of a polarized epithelium.J Cell Biol. 1989; 109: 2817-2832Crossref PubMed Scopus (381) Google Scholar;Gilbert et al., 1991Gilbert T. LeBivic A. Quaroni A. Rodriguez-Boulan E. Microtubular organization and its involvement in the biogenetic pathways of plasma membrane protein in Caco-2 intestinal epithelial cells.J Cell Biol. 1991; 113: 275-288Crossref PubMed Scopus (184) Google Scholar). When these cells are grown to confluency they form specific intercellular junctions that allow demarkation of an apical and a basolateral plasma membrane domain. In parallel, there is a fundamental rearrangement of the microtubule cytoskeleton. In isolated cells, microtubules radiate from a region near the nucleus, containing the centrioles and the Golgi apparatus, towards the cell periphery. In confluent polarized cells, the centrioles have split, migrated under the apical plasma membrane (Buendia et al., 1990Buendia B.M.H. Bré M.H. Griffiths G. Karsenti E. Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.J Cell Biol. 1990; 110: 1123-1135Crossref PubMed Scopus (78) Google Scholar) and the microtubules no longer originate from the pericentriolar region but are arranged, with their plus ends towards the basal membrane, in bundles running parallel to the apico-basal axis and in sparse basal and dense apical networks (Bacallao et al., 1989Bacallao R. Antony C. Dotti C. Karsenti E. Stelzer E.H.K. Simons K. The subcellular organization of Madin-Darby Canine Kidney cells during the formation of a polarized epithelium.J Cell Biol. 1989; 109: 2817-2832Crossref PubMed Scopus (381) Google Scholar;Gilbert et al., 1991Gilbert T. LeBivic A. Quaroni A. Rodriguez-Boulan E. Microtubular organization and its involvement in the biogenetic pathways of plasma membrane protein in Caco-2 intestinal epithelial cells.J Cell Biol. 1991; 113: 275-288Crossref PubMed Scopus (184) Google Scholar). This reorganization of microtubules is concomitant with their stabilization (Bré et al., 1990Bré M.H. Pepperkok R. Hill A.M. Levilliers N. Ansorge W. Selzer E.H.K. Karsenti E. Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells.J Cell Biol. 1990; 111: 3013-3021Crossref PubMed Scopus (92) Google Scholar;Pepperkok et al., 1990Pepperkok R. Bré M.H. Davoust J. Kreis T.E. Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.J Cell Biol. 1990; 111: 3003-3012Crossref PubMed Scopus (75) Google Scholar). Although keratinocytes do not display high levels of apico-basal polarity, rearrangement of their microtubule network has also been observed upon differentiation in vitro (Lewis et al., 1987Lewis L. Barrandon Y. Green H. Albrecht-Buelher G. The reorganization of microtubules and microfilaments in differentiating keratinocytes.Differentiation. 1987; 36: 228-233Crossref PubMed Scopus (17) Google Scholar;Girolomoni et al., 1992Girolomoni G. Stone D.K. Bergstresser P.R. Cruz P.D. Increased number of microtubule-associated dispersal of acidic intracellular compartments accompany differentiation of cultured human keratinocytes.J Invest Dermatol. 1992; 98: 911-917Crossref PubMed Scopus (6) Google Scholar). Microtubule dynamics and organization are presumably controlled by microtubule-associated proteins (MAP) whose expression levels vary with the cell type and differentiation status. MAP were initially identified in neuroneal tissues, because the brain is an important source of microtubules, based on their property to copurify with microtubules during cycles of polymerization and depolymerization. These are MAP1, MAP2, and tau (Matus, 1988Matus A. Microtubule-associated proteins: their potential role in determining neuronal morphology.Ann Rev Neurosci. 1988; 11: 29-44Crossref PubMed Scopus (502) Google Scholar;Wiche, 1989Wiche G. High-Mr microtubule-associated proteins: properties and functions.Biochem J. 1989; 259: 1-12Crossref PubMed Scopus (79) Google Scholar;Maccioni and Cambiazo, 1995Maccioni R.B. Cambiazo V. Role of microtubule-associated proteins in the control of microtubule assembly.Physiol Rev. 1995; 75: 835-864Crossref PubMed Scopus (324) Google Scholar;Mandelkow and Mandelkow, 1995Mandelkow E. Mandelkow E.M. Microtubules and microtubule-associated proteins.Curr Opin Cell Biol. 1995; 7: 72-81Crossref PubMed Scopus (364) Google Scholar). Few non-neuroneal MAP have been characterized so far. MAP4, the best characterized of these MAP, is a family of proteins translated from alternatively spliced RNA that display different tissue distributions (Chapin et al., 1995Chapin S.J. Lue C.M. Yu M.T. Bulinski J.C. Differential expression of alternatively spliced forms of MAP4: a repertoire of structurally different microtubule-binding domains.Biochemistry. 1995; 34: 2289-2301Crossref PubMed Scopus (61) Google Scholar). MAP have at first been shown to modulate the dynamic behavior of microtubules in vitro. Transfection of MAP cDNA into fibroblasts and insect cells has been widely used to demonstrate the role of MAP in the control of microtubule dynamics in cells and for the characterization of these molecules (Lee, 1993Lee G. Non motor MAP.Curr Opin Cell Biol. 1993; 5: 88-94Crossref PubMed Scopus (72) Google Scholar;Hirokawa, 1994Hirokawa N. Microtubule organization and dynamics dependent on microtubule-associated proteins.Curr Opin Cell Biol. 1994; 6: 74-81Crossref PubMed Scopus (338) Google Scholar). E-MAP-115 is a MAP predominantly expressed in epithelial cell lines (Masson and Kreis, 1993Masson D. Kreis T.E. Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.J Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (85) Google Scholar) and in the epithelial component of several organs (Fabre-Jonca et al., 1998Fabre-Jonca N. Allaman J.M. Radlgruber G. Meda P. Kiss J.Z. French L.E. Masson D. The distribution of the murine E-MAP-115 (115 kDa epithelial microtubule-associated protein) during embryogenesis and in adult organs suggests a role in epithelial polarization and differentiation.Differentiation. 1998; 63: 169-180Crossref PubMed Scopus (24) Google Scholar). A role for E-MAP-115 in stabilizing and reorganizing the microtubule cytoskeleton during epithelial cell polarization and differentiation has been proposed based on the observations that: (i) E-MAP-115 expression increases with cell polarization in epithelial cell lines and developing organs; (ii) transient transfection of fibroblasts that do not have detectable levels of E-MAP-115 with the cDNA encoding this protein induces stabilization and reorganization of their microtubules; (iii) the interaction of E-MAP-115 with microtubules decreases at the onset of mitosis when microtubules become more dynamic (Masson and Kreis, 1995Masson D. Kreis T.E. Binding of E-MAP-115 to microtubules is regulated by cell cycle-dependent phosphorylation.J Cell Biol. 1995; 131: 1015-1024Crossref PubMed Scopus (53) Google Scholar). Human epidermis is a stratified epithelium in which basal proliferative keratinocytes progressively stop dividing and enter terminal differentiation as they progress through the suprabasal layers to finally produce a protective outer coat of dead terminally differentiated cells (Fuchs, 1990Fuchs E. Epidermal differentiation: the bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Fuchs and Byrne, 1994Fuchs E. Byrne C. The epidermis: rising to the surface.Curr Opin Genet Develop. 1994; 4: 725-736Crossref PubMed Scopus (218) Google Scholar). Epidermal terminal differentiation can be partially reproduced in vitro using human primary keratinocytes. Keratinocytes grown in low [Ca2+] medium are prevented from stratifying but the onset of terminal differentiation still occurs in about 30%–50% of the cells that display an increased size, express involucrin, and are finally expelled from the basal layer. Raising the Ca2+ concentration in the culture medium triggers terminal differentiation of most keratinocytes; they stop proliferating, develop cellular junctions, form multilayers, and express other terminal gene products such as keratins K1, K10, or filaggrin (Fuchs and Green, 1980Fuchs E. Green H. Change in keratin gene expression during terminal differentiation of the keratinocyte.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (779) Google Scholar;Watt and Green, 1982Watt F.M. Green H. Stratification and terminal differentiation of cultured epidermal cells.Nature. 1982; 295: 434-436Crossref PubMed Scopus (233) Google Scholar;Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33s-40sAbstract Full Text PDF PubMed Scopus (914) Google Scholar;Hennings and Holbrook, 1983Hennings H. Holbrook K.A. Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study.Exp Cell Res. 1983; 143: 127-142Crossref PubMed Scopus (289) Google Scholar;Watt, 1984Watt F.M. Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis.J Cell Biol. 1984; 98: 16-21Crossref PubMed Scopus (135) Google Scholar;Pillai et al., 1990Pillai S. Bikle D.D. Mancianti M.L. Cline P. Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium.J Cell Physiol. 1990; 143: 294-302Crossref PubMed Scopus (193) Google Scholar). We have used human skin and primary keratinocytes to assess a possible function of E-MAP-115 in stabilizing and reorganizing the microtubule cytoskeleton during epithelial cell differentiation. Our results indicate that E-MAP-115 expression correlates with the degree of terminal differentiation of keratinocytes in vivo and in vitro. Interestingly, in primary keratinocytes E-MAP-115 is typically relocalized to stable cortical microtubule bundles facing intercellular contacts early during terminal differentiation. Taken together our results strongly support a role for E-MAP-115 in setting up the basis of the architecture of terminally differentiating keratinocytes. Commercially available monoclonal antibodies directed against actin (MAB1501, Chemicon, Temecula, CA), desmoplakin I and II (DP1 & 2–2.15, Boehringer, Mannheim, Germany), filaggrin (BT-516, BTI, Stoughton, MA), and involucrin (I9018, Sigma, St Louis, MO) were used for immunoblotting and immunofluorescence labeling. Rabbit polyclonal antibodies were used to detect tyrosinated tubulin (anti-T13) and detyrosinated tubulin (anti-T12) (Kreis, 1987Kreis T.E. Microtubules containing detyrosinated tubulin are less dynamic.Embo J. 1987; 6: 2597-2606Crossref PubMed Scopus (311) Google Scholar) and human E-MAP-115 was detected by different polyclonal (R2) or monoclonal (D9C1, D6B2) antibodies (Masson and Kreis, 1993Masson D. Kreis T.E. Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.J Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (85) Google Scholar). Horseradish peroxydase-coupled goat anti-mouse or anti-rabbit IgG (BioRad Labs, München, Germany) and rhodamine- or fluorescein-conjugated anti-mouse or anti-rabbit F(ab′)2 fragment IgG (Jackson Immunoresearch, West Grove, PA) were used as secondary antibodies for immunoblotting and for immunofluorescence labeling, respectively. F-actin was labeled using rhodamine-coupled phalloidin. Nocodazole, taxol, and cytochalasin D stock solutions at 5 mM, 10 mM, or 1 mM, respectively, were prepared in dimethyl sulfoxide and stored at –20°C. Epidermal keratinocytes were isolated from human breast or abdominal skin. Briefly, skin was cut into 0.25 cm2 pieces that were incubated overnight at 4°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 20 mM HEPES pH 7.3, and 1.2 U dispase per ml. The epiderm was then separated from the derm and treated with 0.1% trypsin in phosphate-buffered saline for 20 min at 37°C. Trypsin activity was then inhibited by adding one volume of Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and the cell suspension was filtered, homogeneized, and rinsed twice with the same medium. Primary cells were plated at a density of 7 × 104 per cm2 in FAD culture medium (25% Ham’s F12 medium and 75% Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.13 mM adenine, 0.142 U insulin per ml, 4 nM triiodothyronine, 2 mM hydrocortisone, 292 mg glutamine per ml, 10 ng epidermal growth factor per ml, 0.84 mg cholera toxin per ml, 100 U penicillin per ml, 100 mg streptomycin per ml, 0.25 mg fungizone per ml) on mitomycin growth-arrested human fibroblasts as feeder layers. The medium was changed every 2–3 d until keratinocytes reached about 80%–90% confluency. Monolayers were then either subcultured in defined keratinocyte-SFM medium (GibcoBRL, Basel, Switzerland) containing 0.09 mM Ca2+, or resuspended in freezing medium (10% dimethyl sulfoxide in fetal calf serum) and stored in liquid nitrogen. Cells were routinely grown to 80%–90% confluency at 37°C in an atmosphere of 5% CO2 in SFM and the medium was changed every 2–3 d. Cells used for experiments were always under the third passage and were plated at a density of 12.5 × 102 per cm2 in 100 mm Petri dishes or 24 wells plates containing coverslips that had previously been treated with coating medium (0.01 mg fibronectin per ml, 0.03 mg collagen per ml, 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. Differentiating cells were further grown for up to 10 d. MoAb D9C1 against E-MAP-115 was used for immunocytochemistry on paraffin sections of Bouin’s fixed human epidermis sections using a streptavidin and biotin complex (streptABComplex/HRP; Dako, Glostrup, Denmark) according to the manufacturer’s instructions and 3-amino-9-ethylcarbazole as a chromogen for peroxydase. Keratinocytes were trypsinized, counted, and then lyzed in hot sample buffer and samples corresponding to equivalent numbers of cells were analyzed on 8% polyacrylamide gels according toLaemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202391) Google Scholar, followed by transfer of proteins to nitrocellulose as previously described (Masson and Kreis, 1993Masson D. Kreis T.E. Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.J Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (85) Google Scholar). Immunoblots were developed using an ECL-chemiluminescent detection kit (Amersham, Arlington Heights, IL). Total RNA was extracted from keratinocytes according to the procedure ofChomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62247) Google Scholar, denatured in the presence of glyoxal and dimethyl sulfoxide, electrophoresed through a 1% agarose gel in 10 mM phosphate buffer pH 6.8, and transferred to Hybond-N nylon membrane (Amersham) by capillarity. Prehybridation and hybridation were performed at 65°C in 50% formamide, 50 mM Pipes, 0.8 M NaCl, 2 mM ethylenediamine tetraacetic acid, 2.5 × Denhardt’s, 0.1 mg denatured salmon sperm DNA per ml and 0.1% sodium dodecyl sulfate. cRNA probes were synthesized using α32P-UTP (Amersham) from Bluescript KS plasmids containing either a cDNA fragment coding for a truncated form of human E-MAP-115 (Mb, human E-MAP-115 lacking amino acids 81–426;Masson and Kreis, 1993Masson D. Kreis T.E. Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.J Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (85) Google Scholar) linearized at NheI or the cDNA coding for the 18S RNA. Hybridized RNA were visualized by autoradiography. Keratinocytes grown on glass coverslips were fixed either in methanol for 5 min at –20°C or in 3% paraformaldehyde and processed as described (Masson and Kreis, 1995Masson D. Kreis T.E. Binding of E-MAP-115 to microtubules is regulated by cell cycle-dependent phosphorylation.J Cell Biol. 1995; 131: 1015-1024Crossref PubMed Scopus (53) Google Scholar). In some experiments, cells were fixed in methanol after preextraction in 0.5% Triton X-100, 80 mM K-Pipes pH 6.8, 5 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM MgCl2. Epifluorescence microscopy was performed using a Zeiss 63 × 1.4 planapo oil immersion objective on a Zeiss axiophot microscope (Carl Zeiss, Oberkochen, Germany). Digitized fluorescence images were obtained using a CoolView camera (Photonic Science) coupled to the microscope. In some experiments cells were analyzed using a confocal laser scanning microscope, Zeiss LSM 410 invert, equipped with Ar (488 nm) and HeNe (543 nm) lasers. Immunocytochemical analysis of sections of normal human skin for E-MAP-115 revealed the presence of the protein in the epidermis, whereas it was absent from cells of the dermis (Figure 1a ). Interestingly, higher levels of E-MAP-115 were detected in the terminally differentiating suprabasal than in the proliferative basal layers of keratinocytes. Analysis of sections of basal cell carcinomas and squamous cell carcinomas were done in parallel. The hyperproliferative basal-like keratinocytes of basal cell carcinoma were negative for E-MAP-115, contrary to cells of the suprabasal layers in which E-MAP-115 was still present (Figure 1b). Most of the spreading squamous-like keratinocytes of squamous cell carcinoma were positive for E-MAP-115 (Figure 1c). Strikingly, highest levels of the protein were found in cells of the center of the tumor that are known to be more differentiated (Stoler et al., 1988Stoler A. Kopan R. Duvic M. Fuchs E. Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation.J Cell Biol. 1988; 107: 427-448Crossref PubMed Scopus (285) Google Scholar). Taken together, these observations indicate a correlation between E-MAP-115 expression and the degree of epidermal differentiation in vivo. Terminal differentiation of human primary keratinocytes was induced by raising the Ca2+ concentration in the culture medium and monitored by immunoblotting using involucrin and profilaggrin, as early and late differentiation markers, respectively, for 10 d (Figure 2a ). As observed by others (Rice and Green, 1979Rice R.H. Green H. Presence of human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (620) Google Scholar;Banks-Schlegel and Green, 1981Banks-Schlegel S. Green H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia.J Cell Biol. 1981; 90: 732-737Crossref PubMed Scopus (272) Google Scholar;Watt and Green, 1981Watt F.M. Green H. Involucrin synthesis is correlated with cell size in human epidermal cultures.J Cell Biol. 1981; 90: 738-742Crossref PubMed Scopus (212) Google Scholar;Watt, 1984Watt F.M. Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis.J Cell Biol. 1984; 98: 16-21Crossref PubMed Scopus (135) Google Scholar), involucrin, which is one of the major components of the cornified envelope, could already be detected in keratinocytes grown in low [Ca2+] medium; however, it was ≈2.5 times more abundant after 1 d in high [Ca2+] medium. Profilaggrin, the precursor of the intermediate filament-aggregating protein filaggrin synthesized in cells of the granular layer (Fleckman et al., 1985Fleckman P. Dale B.A. Holbrook K.A. Profilaggrin, a high-molecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes.J Invest Dermatol. 1985; 85: 507-512Abstract Full Text PDF PubMed Scopus (93) Google Scholar;Steven et al., 1990Steven A.C. Bisher M.E. Roop D.R. Steinert P.M. Biosynthetic pathways of filaggrin and loricrin-two major proteins expressed by terminally differentiated epidermal keratinocytes.J Struct Biol. 1990; 104: 150-162Crossref PubMed Scopus (161) Google Scholar), was detected from 3 d of growth in high [Ca2+] medium onwards and levels of this protein continuously increased with time. E-MAP-115 expression was followed in parallel western (Figure 2a) and northern (Figure 2b) blot analysis and the level of the protein and corresponding mRNA quantitated by densitometric scanning (Figure 2c). Interestingly, E-MAP-115 protein was already detected in proliferating keratinocytes but its cellular content was increased 3-fold after 1 d of culture in high [Ca2+] 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 [Ca2+] 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 (Figure 2a). Thus, this analysis clearly demonstrates a quantitative upregulation of E-MAP-115 expression upon keratinocyte terminal differentiation. The intracellular 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 [Ca2+] medium, microtubules formed a dense network surrounding the nucleus and were weakly and discontinuously stained for E-MAP-115 (Figure 3a,b). Raising the Ca2+ concentration in the culture medium triggered the formation of intercellular contacts and, interestingly, microtubules reorganized into bundles directed towards some of these contacts (Figure 3d,f). Concomitantly, E-MAP-115 was completely relocalized to these cortical spike-like microtubule bundles (Figure 3c,e). E-MAP-115 redistribution was an early process because it was observed in some cells already 1 h after raising the calcium concentration (Figure 3c,d) and in all cells after 6 h (Figure 3e,f), when no increase in the levels of this protein could be detected (as monitored by immunoblotting; not shown). Interestingly, as observed in sections of immunolabeled cells obtained by confocal microscopy (Figure 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 [Ca2+] onwards, when stratification had occurred, typical spike-like cortical microtubule bundles could not be observed anymore and microtubules had reorganized into a dense network that was homogeneously labeled for E-MAP-115 (results not shown). Taken together these results hint at a role of E-MAP-115 in the process of stratification.Figure 4E-MAP-115-positive microtubule bundles are restricted to the upper domain of terminally differentiating keratinocytes. Confocal sectioning images of human keratinocytes grown for 6 h in high [Ca2+] medium, fixed in methanol, and stained for E-MAP-115 (red) and tubulin (green) as described in Figure 3 are shown. Optical sectioning was adju" @default.
• W2021287789 created "2016-06-24" @default.
• W2021287789 creator A5014896478 @default.
• W2021287789 creator A5021906809 @default.
• W2021287789 creator A5028235869 @default.
• W2021287789 creator A5057938271 @default.
• W2021287789 date "1999-02-01" @default.
• W2021287789 modified "2023-10-13" @default.
• W2021287789 title "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" @default.
• W2021287789 cites W124166619 @default.
• W2021287789 cites W1573751470 @default.
• W2021287789 cites W1876616931 @default.
• W2021287789 cites W1966975951 @default.
• W2021287789 cites W1969887412 @default.
• W2021287789 cites W1970981016 @default.
• W2021287789 cites W1973869185 @default.
• W2021287789 cites W1974669593 @default.
• W2021287789 cites W1980907691 @default.
• W2021287789 cites W1985091308 @default.
• W2021287789 cites W1985499212 @default.
• W2021287789 cites W1987861844 @default.
• W2021287789 cites W1988711427 @default.
• W2021287789 cites W1989588484 @default.
• W2021287789 cites W1991738124 @default.
• W2021287789 cites W1992480822 @default.
• W2021287789 cites W1993212221 @default.
• W2021287789 cites W1993653794 @default.
• W2021287789 cites W1994060267 @default.
• W2021287789 cites W1994893503 @default.
• W2021287789 cites W1997986612 @default.
• W2021287789 cites W2001062031 @default.
• W2021287789 cites W2001716659 @default.
• W2021287789 cites W2007961473 @default.
• W2021287789 cites W2015171213 @default.
• W2021287789 cites W2020088099 @default.
• W2021287789 cites W2028869988 @default.
• W2021287789 cites W2030776653 @default.
• W2021287789 cites W2039600318 @default.
• W2021287789 cites W2041460044 @default.
• W2021287789 cites W2043909845 @default.
• W2021287789 cites W2045182840 @default.
• W2021287789 cites W2045424274 @default.
• W2021287789 cites W2046560218 @default.
• W2021287789 cites W2061272721 @default.
• W2021287789 cites W2062300120 @default.
• W2021287789 cites W2065019432 @default.
• W2021287789 cites W2069771076 @default.
• W2021287789 cites W2073164746 @default.
• W2021287789 cites W2074790526 @default.
• W2021287789 cites W2076740490 @default.
• W2021287789 cites W2090394023 @default.
• W2021287789 cites W2093374415 @default.
• W2021287789 cites W2094807512 @default.
• W2021287789 cites W2100837269 @default.
• W2021287789 cites W2106792546 @default.
• W2021287789 cites W2107561141 @default.
• W2021287789 cites W2123283133 @default.
• W2021287789 cites W2127012355 @default.
• W2021287789 cites W2140925807 @default.
• W2021287789 cites W2146125957 @default.
• W2021287789 cites W2148004059 @default.
• W2021287789 cites W2151525547 @default.
• W2021287789 cites W2158246711 @default.
• W2021287789 cites W2159671913 @default.
• W2021287789 cites W2173145139 @default.
• W2021287789 cites W2175920363 @default.
• W2021287789 cites W2189417107 @default.
• W2021287789 cites W2189426703 @default.
• W2021287789 cites W4231551516 @default.
• W2021287789 cites W4294216491 @default.
• W2021287789 doi "https://doi.org/10.1046/j.1523-1747.1999.00500.x" @default.
• W2021287789 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9989799" @default.
• W2021287789 hasPublicationYear "1999" @default.
• W2021287789 type Work @default.
• W2021287789 sameAs 2021287789 @default.
• W2021287789 citedByCount "14" @default.
• W2021287789 countsByYear W20212877892012 @default.
• W2021287789 countsByYear W20212877892021 @default.
• W2021287789 countsByYear W20212877892022 @default.
• W2021287789 countsByYear W20212877892023 @default.
• W2021287789 crossrefType "journal-article" @default.
• W2021287789 hasAuthorship W2021287789A5014896478 @default.
• W2021287789 hasAuthorship W2021287789A5021906809 @default.
• W2021287789 hasAuthorship W2021287789A5028235869 @default.
• W2021287789 hasAuthorship W2021287789A5057938271 @default.
• W2021287789 hasBestOaLocation W20212877891 @default.
• W2021287789 hasConcept C104317684 @default.
• W2021287789 hasConcept C127561419 @default.
• W2021287789 hasConcept C17744445 @default.
• W2021287789 hasConcept C185592680 @default.
• W2021287789 hasConcept C199539241 @default.
• W2021287789 hasConcept C20418707 @default.
• W2021287789 hasConcept C55493867 @default.
• W2021287789 hasConcept C74080474 @default.
• W2021287789 hasConcept C79879829 @default.
• W2021287789 hasConcept C86803240 @default.
• W2021287789 hasConcept C94625758 @default.
• W2021287789 hasConcept C95444343 @default.

• next