1 Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
2 Protein Analysis Facility, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
3 Structural Analysis Group, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4 Institute for Physiological Chemistry, Medical Faculty, University of Halle, Magdeburger Strasse 18, 06097 Halle/Saale, Germany
* Author for correspondence (e-mail: w.franke{at}dkfz.de)
Accepted 4 August 2003
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Summary |
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Key words: Lens fibers, Adherens Junctions, Cortex adhaerens, Cadherins, Catenins, Ezrin, Moesin, Periaxin, Periplakin, Desmoyokin
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Introduction |
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It has, however, become increasingly clear over the past decade that there are diverse adhering junctions that cannot be subsumed under these two major categories but represent special structures sui generis. Examples include the complexus adhaerentes described in special vascular endothelia, notably the retothelial cells of lymph node sinus (Schmelz and Franke, 1993; Valiron et al., 1996
), the M-/N-cadherin containing contactus adhaerentes connecting the cells of the granule layer of the cerebellum (Rose et al., 1995
; Hollnagel et al., 2002
), the area composita in the intercalated disks connecting cardiomyocytes (e.g. C. M. Borrmann, Molekulare Charakterisierung der Adhärens-Zellverbindungen des Herzens: Identifizierung einer neuen Art, der Area composita, PhD Thesis, University of Heidelberg, Germany, 2000) and the heterotypic adhering junctions connecting the photoreceptor and Mueller glia cells of the retina, which characteristically contain the arm-protein neurojungin (Paffenholz et al., 1999
).
The eye lens contains a central mass of densely packed anucleate fiber cells surrounded by a layer of cells with epithelioid features, often referred to as `epithelium'; this layer is in turn surrounded by a capsule of extracellular matrix material (for reviews, see Maisel et al., 1981; Rafferty, 1985
). The physical laws of the vision process require transparency and homogeneity of the lens body. Therefore, the tight package of the lens fibers, with frequent and often regularly spaced interdigitations, as well as the high concentration and homogenous distribution of cytoplasmic proteins, including loosely arranged cytoskeletal filaments, are crucial for lens function (Ramaekers et al., 1980
; Benedetti et al., 1981
; Maisel et al., 1981
; Ramaekers and Bloemendal, 1981
). It is thus not surprising that diverse disturbances of the composition and distribution of lens proteins all lead to cataract formation (e.g. Capetanaki et al., 1989
; Duncan et al., 2000
; He and Li, 2000
; Jakobs et al., 2000
; Krutovskikh and Yamasaki, 2000
).
Previously, we have noted that the cell-cell interactive structure of the lens fibers represents a relatively thin but extended cortex around the entire cell, obviously a cell-type-specific junctional complex (Schmidt et al., 1994). This cortex comprises the plasma membrane proper and a subjacent plaque-equivalent layer that only rarely shows distinct substructures (cf. Franke et al., 1987
; Lo et al., 1997
; Lo et al., 2000
) but is generally associated with actin microfilaments, actin-binding proteins (ABPs) and adhering junction proteins including plakoglobin (Franke et al., 1987
),
- and ß-catenin (Bassnett et al., 1999
; Duncan et al., 2000
; Bagchi et al., 2002
),
-actinin and vinculin (Geiger et al., 1985
; Beebe et al., 2001
). N-Cadherin has been reported to be the most prominent, if not the only, cadherin present (e.g. Hatta and Takeichi, 1986
; Atreya et al., 1989
; Citi et al., 1994
; Lo et al., 2000
; Bagchi et al., 2002
). More recently, however, we have noticed that this cortex is highly complex and heterogenous, and comprises a set of proteins hitherto not shown or even expected in the lens.
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Materials and Methods |
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Antibodies and reagents
Antibodies used included mouse monoclonal antibodies (mAbs) against: ezrin (3C12), N-cadherin, vinculin (hvin-1), actin and tropomyosin from Sigma (St Louis, MO, USA); fodrin/spectrin (MAB 1822) from Chemicon (Hofheim/Taunus, Germany); drebrin from MoBiTec (Göttingen, Germany); E-, N-, P- and R-cadherin, cadherin-5, moesin, - and ß-catenin, and p120ctn from Transduction Laboratories (Lexington, KY, USA); protein p0071 (mAb 6D-1-10) (Hatzfeld et al., 2003
); and cadherin-11 from Zymed (South San Francisco, CA, USA). In addition, we used a series of mAbs from Progen Biotechnik (Heidelberg, Germany): desmoplakins (DP I/II, 2.15, 2.17 and 2.20) (Cowin et al., 1985
), desmogleins Dsg1-Dsg3 (e.g. Dsg 1&2: 3.10.), desmocollins Dsc1-Dsc3 (e.g. mAbs U100 and U114), plakoglobin [Pg 5.10 (Cowin et al., 1986
); 11E4], vimentin (3B4 and V9) (Hermann et al., 1989), plakophilins PKP1-PKP3 (Mertens et al., 1996
; Schmidt et al., 1999
) and an antibody to neurojungin (J 19.97) that in lens tissue also reacts with phakinin (cf. Paffenholz et al., 1999
). Rabbit antibodies routinely used were against
-catenin, ß-catenin, pan-cadherin, tropomyosin,
-actinin, l/s-afadin, l-afadin (Sigma) (for a review, see Takai and Nakanishi, 2003
), protein ZO-1, connexin Cx 43, ponsin, claudin-1, occludin, cadherin-11 (from Zymed), non-muscle myosin heavy chain (Biotrend, Cologne, Germany), merlin/NF-2 from Santa Cruz Biotechnology (Heidelberg, Germany), CD44 (generous gift from M. Zöller, German Cancer Research Center) and protein p0071 (Hatzfeld et al., 2003
). Specific guinea-pig antisera against plectin were also used (P2, from H. Herrmann, German Cancer Research Center) (cf. Schröder et al., 1999
).
Monoclonal antibodies against desmoyokin (Dy 2.4. and Dy 47.27.5) obtained in this laboratory were systematically compared with desmoyokin rabbit antisera kindly provided by T. Hashimoto (Department of Dermatology, Keio University of Medicine, Tokyo, Japan) (cf. Hashimoto et al., 1993). To generate further antibodies specific for human desmoyokin, the synthetic peptides D1 (amino acids 2038-2056: PDVKIPKFKKPKFGFGPKS; `AHNAK fragment', accession number A45259), D2 (amino acids 2792-2812: PKGKGGVTGSPEASISGSKGD) and D4 (amino acids 298-320: PNLEGTLTGPRLGSPSGKTGT; all peptides used were from Peptide Specialty Laboratories, Heidelberg, Germany) were coupled to KLH and used to immunize guinea pigs after dissolution in Freund's complete adjuvant (Sigma). After three booster injections using Freund's incomplete adjuvant, the animals were anesthetized and blood was collected by heart puncture.
Periplakin antibodies used included murine mAbs [AE11 (Ma and Sun, 1986); clone IIb (Simon and Green, 1984
); generous gifts from T. T. Sun (Department of Dermatology, New York University Medical Center, NY) and M. Simon (School of Dental Medicine, SUNY, Stony Brooks, NY, USA)] as well as guinea pig antibodies generated against three synthetic peptides, P1 (amino acids 7-24: KRNKGKYSPTVQTRSISN; accession number AAC 17738), P2 (amino acids 336-356: LRKVDSDLNQKYGPDFKDRYQ) and P3 (amino acids 815-835: ENGRSSHVSKRARLQSPATKV) as described above for desmoyokin.
Immunohistochemistry
Lens cryosections of 5 µm thickness were air dried for several hours and fixed for 5 minutes in methanol, followed by 5 minutes in acetone both at 20°C. Because of the fragile character of lens, the incubation protocols were optimized for relatively brief periods of incubation time, and the specimens were usually incubated with the primary antibodies diluted in PBS for 30 minutes, followed by two repeated washes in PBS for 5 minutes or less each, incubation with the secondary antibodies for 30 minutes, and two subsequent 5-minute washes in PBS. The sections were then rinsed in distilled water, fixed for 5 minutes in ethanol and mounted in Fluoromount G (Biozol Diagnostica, Eching, Germany). To enhance the accessibility of certain large proteins such as desmoyokin and periplakin to immunoglobulins, the specimens were initially exposed to PBS containing either 0.1% Triton X-100 or 0.1% saponin and then washed twice in PBS for 5 minutes each. Immunofluorescence of cell cultures and of other tissues was performed as described (Peitsch et al., 1999
).
Epifluorescence was observed and documented with a Zeiss Axiophot photomicroscope, confocal laser-scanning immunofluorescence microscopy was done with a Zeiss LSM 510 (Zeiss, Jena, Germany).
Fractionation of tissues and cell cultures
Cortex preparations from several lenses were homogenized with either a Dounce or a Potter-Elvehjem homogenizer (Braun, Melsungen, Germany) at very high volume-to-tissue mass ratios in low salt buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM DTT) containing the protease inhibitor phenylmethylsulfonylfluoride (PMSF) at 1 mM or Pefablock SC (Roche Diagnostics, Mannheim, Germany). Pellets obtained after centrifugation at 4°C at 18,000 g (in a Beckman centrifuge Optima XL-70, München, Germany) for 30 minutes contained the `water-insoluble' particle fraction (WIF), primarily the cytoskeleton and membranous structures, whereas the supernatant contained the water-soluble particle fraction (WSF) including the crystallins (Alcala et al., 1975). This procedure was repeated twice with the WIF to minimize residual crystallins. Tissues other than lens and cell cultures were directly homogenized in SDS sample buffer as described (Peitsch et al., 1999
).
Gel electrophoresis and immunoblotting SDS-PAGE, and two-dimensional gel electrophoresis involving either non-equilibrium pH-gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) were performed as described (Achtstätter et al., 1986). For SDS-PAGE, samples were suspended in electrophoresis buffer (250 mM Tris-HCl, pH 6.8, 20% SDS, 25% glycerol, 125 mM DTT), often with the addition of benzonase (1:1000; Merck, Darmstadt, Germany). For NEPHGE or IEF, protein samples were precipitated with methanol and chloroform, and solubilized in lysis buffer containing 9.5 M urea, 2.0% NP-40, 2.0% ampholine and 20 mM DTT.
Immunoblotting was performed using PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 10% non-fat dry milk in Tris-buffered saline containing 0.1% Tween (TBST) for at least 1 hour, blots were incubated with the primary antibodies in PBS for 1 hour, followed by three washes in TBST for 30 minutes each. Horseradish peroxidase (HRP)-conjugated antibodies to rabbit, mouse or guinea pig IgG (diluted 1:10000 in TBST) were applied for 30 minutes, followed by a 30 minute wash in TBST and enhanced chemiluminescence (ECL; Amersham Biosciences, Freiburg, Germany).
Immunoprecipitations
Pelleted fractions were suspended in immunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA or 0.5 mM CaCl2, 1% Triton X-100, 1 mM DTT, 1 mM PMSF or Pefablock SC) and centrifuged for 15 minutes at 14,900 g (in an Eppendorf centrifuge 5414, Hamburg, Germany) and 4°C. The supernatant obtained was then precleared with protein-A- or protein-G-coupled Sepharose for several hours, and the supernatant obtained after centrifugation was reacted overnight with protein A and/or protein G beads coated with the specific antibody in 50 mM Tris-HCl, pH 7.5. The pellet obtained was solubilized in 20-40 µl sample buffer, and the immunoprecipitate was separated using SDS-PAGE. Protein gels stained with colloidal Coomassie Blue (Novex, Frankfurt, Germany) were used to analyse unknown bands by peptide mass fingerprinting [matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)] and amino acid sequence analysis (see below).
Tryptic digestion
Protein bands were excised from the gel and cut into 1x1 mm pieces that were washed twice with deionized water, 50% acetonitrile/water (1:1) and acetonitrile. Proteins were digested overnight with sequencing-grade modified trypsin (Promega) in 40 mM ammonium bicarbonate at 37°C. The reaction was stopped by freezing.
MALDI-MS
MALDI mass spectra were recorded in positive ion reflector mode with delayed extraction on a Reflex II time-of-flight instrument (Bruker-Daltonik, Bremen, Germany) equipped with a SCOUT multiprobe inlet and a 337-nm nitrogen laser. The ion-acceleration voltage was set to 20.0 kV, the reflector voltage to 21.5 kV and the first extraction plate to 15.4 kV. Mass spectra were obtained by averaging 50-200 individual laser shots. Calibration of the spectra was performed internally by a two-point linear fit using the autolysis products of trypsin at mass:charge ratios of 842.50 and 2211.10.
For the mass spectrometric analysis of tryptic digests, MALDI samples were prepared on thin film spots (Jensen et al., 1996). Briefly, 0.3 µl aliquots of a nitrocellulose-containing saturated solution of
-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in acetone were deposited onto individual spots on the target. Subsequently, 0.8 µl 10% formic acid and 0.4 µl of the digest sample were loaded on top of the thin film spots and allowed to dry slowly at ambient temperature. To remove salts from the digestion buffer, the spots were washed with 1% formic acid and with water.
Post-source-decay analysis
Post-source-decay (PSD) analysis was performed in positive ion reflector mode with delayed extraction by setting an ion gate width of 40 Da around the ion of interest. Data were acquired in 14 segments by decreasing the reflector voltage in a stepwise fashion. For each segment, 200 individual laser shots were accumulated. The fragment ion spectrum was obtained by pasting together all segments to a single spectrum using the FAST software (Bruker). Fragment ion calibration was performed externally with the fragment masses of the adrenocorticotropic hormone (ACTH) 18-39 clip.
Sample preparation for PSD analysis was achieved by cocrystallization of matrix with samples concentrated using Zip Tip C18 (Millipore, Schwalbach, Germany). Briefly, the peptides in the supernatant of the in-gel digestion were absorbed to a prewashed (50% acetonitrile/water) and equilibrated (0.1% trifluoroacetic acid/water) Zip Tip C18 by repetitive pipetting steps. Following washing of the Zip Tip C18 by equilibration buffer, the peptides were eluted from the Zip Tip with 1 µl of matrix (-cyano-4-hydroxycinnamic acid saturated in 50% acetonitrile/water).
Database search
Singly charged monoisotopic peptide masses were used for database searching. Searches were performed against the NCBInr database using the ProFound search algorithm (http://129.85.19.192/prowl-cgi/ProFound.exe) and the Protein prospector software developed at the University of California, San Francisco (http://prospector.ucsf.edu/), with an IEP range of 0-14 and the oxidation of methionine as a possible modification. Up to one missed tryptic cleavage was considered, and the mass tolerance for the monoisotopic peptide masses was set to ±100 ppm or ±0.1 Da.
Searches with fragment masses from PSD experiments were performed against the NCBInr database using the MS-Tag search algorithm provided by Protein prospector. Parent mass tolerance was set to ±100 ppm and fragment ion tolerance was set to ±1500 ppm.
Amino acid sequence analysis
For high-performance liquid chromatography (HPLC) separation the tryptic digest was extracted twice with 0.1% trifluoroacetic acid (TFA) in 60% acetonitrile. After concentration on a SpeedVac, the extracted tryptic peptides were separated on a HPLC system equipped with a 140B solvent delivery system (Applied Biosystems, Weiterstadt, Germany), Acurate splitter (LC Packings, Idstein, Germany), UV absorbance detector 759A (Applied Biosystems), U-Z capillary flow cell (LC Packings) and a Probot fraction collector (BAI, Lautertal, Germany) using a reversed-phase column (Hypersil C18 BDS, mean particle diameter 3 µm, 0.3x150 mm) and a linear gradient from 12% acetonitrile in 0.1%TFA to 64% acetonitrile in 0.08% TFA in 90 minutes, with a flow rate of 4 µl minute1 at room temperature.
Peptide elution was monitored at 214 nm and individual fractions from the HPLC separation were re-analysed by MALDI-MS. Sequence analysis of selected peptide-containing fractions was performed on a Procise Protein Sequencer 494 cLC using standard programs supplied by Applied Biosystems.
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Results |
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Biochemical analysis
When total proteins from mammalian lens fiber tissue, without the capsule and the outermost `epithelioid' cell layer, were extracted with relatively large amounts of `low salt buffer', a residual (`cytoskeletal') fraction was obtained that revealed, on SDS-PAGE or two-dimensional gel electrophoresis, a remarkably complex protein pattern. In addition to the IF proteins vimentin, phakinin and filensin (e.g. Lieska et al., 1980; Ramaekers et al., 1980
; Merdes et al., 1991
; Merdes et al., 1993
; Perng et al., 1999
) (U. Haus, Zur molekularbiologischen Charakterisierung von Cytoskelett-Proteinen der Rinderlinse. Diploma Thesis, University of Cologne, Germany, 1990), some typical junctional proteins were consistently seen in all three species studied. These included the transmembrane glycoproteins N-cadherin, cadherin-11 and in much lower amounts E-cadherin (Fig. 1, lanes 1-6), which seemed to be restricted to the outer layers, as well as the plaque proteins
- and ß-catenin (Fig. 1, lanes 7-10), p120ctn (see below) and, in amounts markedly differing in different species, plakoglobin. Although the finding of an N-cadherin-based ensemble of adhering junction proteins essentially confirmed earlier reports (e.g. Geiger et al., 1985
; Cowin et al., 1986
; Hatta and Takeichi, 1986
; Franke et al., 1987
; Atreya et al., 1989
; Bassnett et al., 1999
; Ferreira-Cornwell et al., 2000
; Leong et al., 2000
; Bagchi et al., 2002
), the recognition that the cortices adhaerentes of lens fibers contained the type-II cadherin-11 in similar large amounts was novel. On the one hand, it was compatible with the general widespread occurrence of cadherin-11 in diverse kinds of mesenchymal and other mesodermally derived cells but, on the other hand, it was surprising because it had not been detected in previous studies of lens tissue (e.g. Hoffmann and Balling, 1995
; Simonneau et al., 1995
; Hadeball et al., 1998
; Simonneau and Thiery, 1998
). The other cadherins examined (see Materials and Methods) were not detected. Among the cytoskeletal proteins, we regularly detected vinculin (Fig. 1, lanes 15 and 16),
-actinin, actin, tropomyosin, myosin, spectrin, ankyrin and plectin (not shown), all of which had previously been reported to occur in lens-fiber cortices (e.g. Kibbelaar et al., 1979
; Repasky et al., 1982
; Allen et al., 1987
; Franke et al., 1987
; Weitzer and Wiche, 1987
; Lee et al., 2000
).
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Much to our surprise, however, we also noted among the major lens junction proteins a series of plaque components such as ezrin (Fig. 1, lanes 11 and 12), periplakin (Fig. 1, lanes 13 and 14), periaxin (see also below) and desmoyokin (Figs 2, 4), which so far had only been reported from other kinds of cells [ezrin (Bretscher, 1983; Bretscher et al., 1997
); periplakin (Simon and Green, 1984
); periaxin (Gillespie et al., 1994
); desmoyokin (cf. Hieda et al., 1989
; Shtivelman et al., 1992
; Hashimoto et al., 1993
)]. In addition, we found considerable amounts of moesin in our immunoblots of total lens fiber proteins, but no significant signals for merlin [see below, however, for reports of the occurrence of merlin in outer (i.e. epithelioid) cells of lenses] (see Claudio et al., 1995
; Claudio et al., 1997
; Huynh et al., 1996
).
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To identify the protein complexes containing these lens cortex proteins, we performed immunoprecipitations, subjected the pelleted proteins to SDS-PAGE, excised the bands under question and analysed them by MALDI-MS, PSD and amino acid sequencing. The results allowed us to determine the complement of proteins associated with the specific antigen. For example, N-cadherin immunoprecipitates consistently contained not only the associated plaque proteins - and ß-catenin, plakoglobin and p120ctn, but also remarkable amounts of cadherin-11 (Fig. 3). Conversely, considerable proportions of N-cadherin were identified in the immunoprecipitates obtained with antibodies to cadherin-11. In lens tissue material also containing outer cortical cell layers, junctional plaque proteins were detected in combinations with both N- and E-cadherin as well as with cadherin-11 (data not shown). These results also showed for the first time the existence of such heterotypic cadherin complexes with common plaque proteins but did not yet allow to distinguish between lateral heterocomplexes in the same membrane from transcellular heterotypic complexes of cadherins (e.g. Volk et al., 1987
); that is, from the `heterocadherins' in the sense used by Duguay et al. (Duguay et al., 2003
) [for the controversial literature on cadherin organization see Shapiro et al., and others (Shapiro et al., 1995
; Leckband and Sivasankar, 2000
; Boggon et al., 2002
; Ahrens et al., 2003
)]. By contrast, ezrin immunoprecipitates contained desmoyokin, periplakin and periaxin (Table 1; Fig. 4a), and, when further probed with specific antibodies, positive reactions were also seen for spectrin (Fig. 4b), plectin and moesin (data not shown). These results left no doubt that desmoyokin was a major protein of lens fiber cells, where it mostly occurred in ezrin complexes, apparently together with periplakin and periaxin (Fig. 4a), suggesting the existence of a special category of large EPPD plaque complexes. For reasons not yet clarified, however, we detected little if any ezrin and moesin in the reciprocal immunoprecipitates of desmoyokin and periplakin (data not shown).
|
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In similar biochemical experiments, we failed to detect in lens fibers significant amounts of desmosome-specific proteins such as desmoplakins, desmogleins, desmocollins and plakophilins, or of the plaque proteins neurojungin, merlin, afadin, ponsin and drebrin (results not shown). The negative results obtained for the transmembrane glycoprotein CD44 were surprising because this protein has been reported in previous studies of lens material by other authors (Nishi et al., 1997; Saika et al., 1998
), but we have not yet excluded all the diverse CD44 variants.
Immunohistochemistry
Neither in our electron micrographs nor by immunofluorescence microscopy did we find, in sections through lens fibers, any indication of the presence of tight junctions, including negative reactions for occludin and several claudins (cf. Langbein et al., 2002), and of desmosomes (see also Franke et al., 1987
). By contrast, various sizes of gap junctions were regularly found by electron microscopy and with antibodies to both connexins and protein ZO-1, especially in the central region of the long cell sides, confirming previous reports (Giepmans and Molenaar, 1998
; Toyofuku et al., 1998
; Nielsen et al., 2001
). The cytoplasm displayed the notorious intense positivity for IF proteins such as vimentin (cf. Ramaekers et al., 1980
) and phakinin (e.g. Fig. 6B) (cf. Merdes et al., 1991
; Merdes et al., 1993
), whereas actin and the ABPs examined appeared to be generally enriched in the cortical zone (cf. Kibbelaar et al., 1979
; Lo, 1988
; Lo et al., 1997
).
|
Using immunohistochemistry, N-cadherin and cadherin-11 were the only cadherins that consistently reacted at the contacts of lens fiber cells, very intensely at the short apical sides and rather weakly, sometimes hardly visible at all, along the long lateral sides (Fig. 5A-C). By contrast, immunostaining for E-cadherin was weakly present in the outer cortical layers but diminished centripetally in a steep gradient (not shown). Particularly at the short sides, both N-cadherin and cadherin-11 colocalized with both -catenin and the major arm-repeat proteins ß-catenin (Fig. 5A-C''), plakoglobin and p120ctn (results not shown), as well as with actin and vinculin (cf. Volk and Geiger, 1984
; Geiger et al., 1985
; Franke et al., 1987
). Specifically in bovine lens, the plakoglobin reaction was relatively strong in the outermost cell layers, but was practically undetectable in the lens interior [for other species, see Franke et al. (Franke et al., 1987
)]. Again, the immunoreaction of all these proteins was very intense at the short sides and weak on the long sides.
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The immunofluorescence reaction pattern of merlin [another cortical protein related to ezrin and moesin (for a review, see Bretscher et al., 2002)] was surprising in two ways. It was generally weak, often negative, in the more cortical regions of the lens fiber mass and stronger in the deeper regions, opposite to what has been reported for mouse and chicken lens (e.g. Claudio et al., 1995
; Claudio et al., 1997
; Huynh et al., 1996
); however, where positive, it often appeared with nearly equal intensity on both the long and the short sides.
Immunolocalization reactions for neurojungin, afadins, ponsin, drebrin and protein CD44 were negative.
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Discussion |
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Taken together, the present results show that the cortex adhaerens hexagons, notably the short sides, are characterized by a typical adhaerens junction ensemble, dominated by N-cadherin and cadherin-11 as major transmembrane glycoproteins and a plaque comprising not only - and ß-catenin, but also plakoglobin, p120ctn and vinculin. The results of our N-cadherin/cadherin-11 cross-immunoprecipitation experiments have also directly demonstrated intimate complexes of different type-I and type-II cadherins with the plaque proteins mentioned, and experiments are under way to decide whether these are ipso- or heterocellular cadherin complexes. The immunocytochemical results further suggest that the long-side cortex of the fiber cells, at least in the species examined, contains much less of the adherens junction components but is relatively rich in ABPs characteristic of other microfilament-anchorage complexes, including band 4 proteins (Aster et al., 1984
; Allen et al., 1987
), spectrin (Nelson et al., 1983
; Green and Maisel, 1984
; Thomas, 2001
) and plectin (Weitzer and Wiche, 1987
).
Our surprising finding of a totally novel group of actin filament anchorage proteins in the lens now adds another ensemble of cytoskeletal proteins to the cortex adhaerens that have hitherto been reported only from diverse other cells. These include ezrin and moesin [cortical ABPs of various epithelial and certain non-epithelial cells (e.g. Bretscher, 1983; Bretscher et al., 1997
; Yonemura et al., 1998
; Bretscher et al., 2002
)], periplakin [a desmoplakin-related protein so far found only in epidermis and other stratified epithelia (Ruhrberg et al., 1997
; DiColandrea et al., 2000
; Karashima and Watt, 2002
) [for periplakin gene transcripts in certain other cells, see also Aho et al. (Aho et al., 1998
)] and desmoyokin [a large protein with a hotly debated location (e.g. Hieda et al., 1989
; Shtivelman et al., 1992
; Hashimoto et al., 1993
; Shtivelman and Bishop, 1993
; Masanuga et al., 1995
)]. In addition, in such complexes, we have detected periaxin, a protein originally identified in myelinating Schwann cells, where it is enriched at plasma membranes (e.g. Gillespie et al., 1994
; Melendez-Vasquez et al., 2001
). Our immunoprecipitation results have further shown that lens-fiber ezrin occurs in the same junctional plaque complexes as periplakin, periaxin and desmoyokin, often also in association with lens spectrin(s), which suggests but does not yet prove that all these proteins can co-assemble into a giant cortical EPPD complex.
The constitutive occurrence of desmoyokin and periaxin in the cortex adhaerens of lens fibers is especially noteworthy because both proteins have been reported as `dual location proteins' that occur in certain plasma membrane regions as well as in the nucleoplasm of a broad variety of cell types (e.g. Shtivelman et al., 1992; Shtivelman and Bishop, 1993
; Masanuga et al., 1995
; Sherman and Brophy, 1999
; Nie et al., 2000
; Sussmann et al., 2001
). The mere absence of nuclei in the lens fiber cells now also demonstrates that desmoyokin and periaxin are indeed major, stable components of adhering junctions that can occur in a long-lasting form in the absence of a nucleus. In this context, it is also worth mentioning that desmoyokin has recently been shown in the area composita plaques of cardiac intercalated disks (e.g. Hohaus et al., 2002
).
At present, the transmembrane protein(s) to which EPPD complexes are attached are still elusive. Although we cannot formally exclude some contribution of cadherins to these cortical complexes, their relatively low concentrations along the long sides of the fiber cell cortices (see Results) (Bassnett et al., 1999; Lo et al., 2000
; Beebe et al., 2001
) and their absence in our EPPD immunoprecipitates tend to suggest an involvement of other transmembrane proteins. Because the known ezrin-binding protein CD44 (Tsukita et al., 1994
; Yonemura et al., 1998
) has not been detected in our ezrin immunoprecipitates, we will have systematically to examine the series of known candidates of possible transmembrane partners in the EPPD complex (for a review, see Bretscher et al., 2002
). It will also be interesting to investigate the possible existence of junctional plaque complexes of the EPPD category in other cell types.
Our high-resolution double-label immunofluorescence microscopy also revealed a mosaicism of the cortex adhaerens, in particular on the short sides of hexagons, where puncta-adhaerentia-type complexes comprising cadherins and catenins alternate with junctional structures containing EPPD complexes. Whether this regular patchwork pattern is a general characteristic of the junction system and how it is formed in the development of lens fibers from the `epithelioid' cells of the lens surface, remain to be studied. Considering the frequency and sensitivity with which alterations of protein composition of lens fibers result in cataract formations, including changes of junctional components such as gap junction connexins (Martinez-Wittingham et al., 2003), we expect that gene abrogation experiments will probably help to elucidate the functional importance of the molecular complexity and pattern arrangement of the cortex adhaerens.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Achtstätter, T., Hatzfeld, M., Quinlan, R. A., Parmelee, D. C. and Franke, W. W. (1986). Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol. 134, 355-371.[Medline]
Aho, S., McLean, W. H. I., Li, K. and Uitto, J. (1998). cDNA cloning, mRNA expression, and chromosomal mapping of human and mouse periplakin genes. Genomics 48, 242-247.[CrossRef][Medline]
Ahrens, T., Lambert, M., Pertz, O., Sasaki, T., Schulthess, T., Mège, R.-M., Timpl, R. and Engel, J. (2003). Homoassociation of VE-cadherin follows a mechanism common to `classical' cadherins. J. Mol. Biol. 325, 733-742.[CrossRef][Medline]
Alcala, J., Lieska, N. and Maisel, H. (1975). Protein composition of bovine lens cortical fiber cell membranes. Exp. Eye Res. 21, 581-595.[Medline]
Allen, D. P., Low, P. S., Dola, A. and Maisel, H. (1987). Band 3 and ankyrin homologues are present in eye lens: evidence for all major erythrocyte membrane components in same non-erythroid cell. Biochem. Biophys. Res. Commun. 149, 266-275.[Medline]
Aster, J. C., Brewer, G. J., Hanashi, M. and Maisel, H. (1984). Band 4.1-like proteins of the bovine lens. Biochem. J. 224, 609-616.[Medline]
Atreya, P. L., Barnes, J., Katar, M., Alcala, J. and Maisel, H. (1989). N-cadherin of the human lens. Curr. Eye Res. 8, 947-956.[Medline]
Bagchi, M., Katar, M., Lewis, J. and Maisel, H. (2002). Associated proteins of lens adherens junction. J. Cell. Biochem. 86, 700-703.[CrossRef][Medline]
Bassnett, S., Missey, H. and Vucemilo, I. (1999). Molecular architecture of the lens fiber cell basal membrane complex. J. Cell Sci. 112, 2155-2165.
Beebe, D. C., Vasiliev, O., Guo, J., Shui, Y.-B. and Bassnett, S. (2001). Changes in adhesion complexes define stages in the differentiation of lens fiber cells. Invest. Ophthalmol. Vis. Sci. 42, 727-734.
Benedetti, E. L., Dunia, I., Ramaekers, F. C. S. and Kibbelaar, M. A. (1981). Lenticular plasma membranes and cytoskeleton. In The Ocular Lens: Structure, Function and Pathology (ed. H. Maisel), pp. 137-188. New York: Marcel Dekker.
Boggon, T. J., Muray, J., Chappuis-Flament, S., Wong, E., Gumbiner, B. M. and Shapiro, L. (2002). C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296, 1308-1313.
Bretscher, A. (1983). Purification of an 80,000 Dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J. Cell Biol. 97, 425-432.[Abstract]
Bretscher, A., Reczek, D. and Berryman, M. (1997). Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J. Cell Sci. 110, 3011-3018.
Bretscher, A., Edwards, K. and Fehon, R. G. (2002). ERM proteins and merlin: integrators at the cell cortex. Nature 3, 586-599.[CrossRef]
Capetanaki, Y., Smith, S. and Heath, J. P. (1989). Overexpression of the vimentin gene in transgenic mice inhibits normal lens cell differentiation. J. Cell Biol. 109, 1653-1664.[Abstract]
Citi, S., Volberg, T., Bershadsky, A. D., Denisenko, N. and Geiger, B. (1994). Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7. J. Cell Sci. 107, 683-692.
Claudio, J. O., Lutchman, M. and Rouleau, G. A. (1995). Widespread but cell type-specific expression of the mouse neurofibromatosis type 2 gene. Neuroreport 6, 1942-1946.[Medline]
Claudio, J. O., Veneziale, R. W., Menko, A. S. and Rouleau, G. A. (1997). Expression of schwannomin in lens and Schwann cells. Neuroreport 27, 2025-2030.
Cowin, P., Kapprell, H.-P. and Franke, W. W. (1985). The complement of desmosomal plaque proteins in different cell types. J. Cell Biol. 101, 1442-1454.[Abstract]
Cowin, P., Kapprell, H.-P., Franke, W. W., Tamkun, J. and Hynes, R. O. (1986). Plakoglobin: a protein common to different kinds of intercellular adhering junctions. Cell 46, 1063-1073.[Medline]
DiColandrea, T., Karashima, T., Määttä, A. and Watt, F. M. (2000). Subcellular distribution of envoplakin and periplakin: insights into their role as precursors of the epidermal cornified envelope. J. Cell Biol. 151, 573-585.
Duguay, D., Ramsey, A. F. and Steinberg, M. S. (2003). Cadherin-mediated cell adhesion and tissue segregation: qualitative and quantitative determinants. Dev. Biol. 253, 309-323.[CrossRef][Medline]
Duncan, M. K., Kozmik, Z., Cveklova, K., Piatigorsky, J. and Cvekl, A. (2000). Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of 5ß1 integrin expression. J. Cell Sci. 113, 3173-3185.
Farquhar, M. and Palade, G. E. (1963). Junctional complexes in various epithelia. J. Cell Biol. 17, 375-412.
Ferreira-Cornwell, M. C., Veneziale, R. W., Grunwald, G. B. and Menko, A. S. (2000). N-Cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro. Exp. Cell Res. 256, 237-247.[CrossRef][Medline]
Franke, W. W., Schmid, E., Grund, C., Müller, H., Engelbrecht, I., Moll, R., Stadler, J. and Jarasch, E.-D. (1981). Antibodies to high molecular weight polypeptides of desmosomes: specific localization of a class of junctional proteins in cells and tissues. Differentiation 20, 217-241.[Medline]
Franke, W. W., Kapprell, H.-P. and Cowin, P. (1987). Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells. Eur. J. Cell Biol. 43, 301-315.[Medline]
Geiger, B., Volk, T. and Volberg, T. (1985). Molecular heterogeneity of adherens junctions. J. Cell Biol. 101, 1523-1531.[Abstract]
Giepmans, B. N. and Moolenaar, W. H. (1998). The gap junction protein connexin 43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr. Biol. 140, 1199-1209.
Gillespie, C. S., Sherman, D. L., Blair, G. E. and Brophy, P. J. (1994). Periaxin, a novel protein of myelinating Schwann cells with a possible role in axonal ensheathment. Neuron 12, 497-508.[Medline]
Green, J. and Maisel, H. (1984). Lens fodrin binds actin and calmodulin. Curr. Eye Res. 3, 1433-1440.[Medline]
Green, K. J. and Gaudry, C. A. (2000). Are desmosomes more than tethers for intermediate filaments? Nat. Rev. Mol. Cell Biol. 1, 208-216.[CrossRef][Medline]
Hadeball, B., Borchers, A. and Wedlich, D. (1998). Xenopus cadherin-11 (Xcadherin-11) expression requires the Wg/Wnt signal. Mech. Dev. 72, 101-113.[CrossRef][Medline]
Hashimoto, T., Amagai, M., Parry, D. A., Dixon, T. W., Tsukita, S., Tsukita, S., Miki, K., Sakai, K., Inokuchi, Y., Kudoh, J. et al. (1993). Desmoyokin, a 630 kDa keratinocyte plasma membrane-associated protein, is a homologous to the protein encoded by human gene AHNAK. J. Cell Sci. 105, 275-286.
Hatta, H. and Takeichi, M. (1986). Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320, 447-449.[Medline]
Hatzfeld, M. (1999). The armadillo family of structural proteins. Int. Rev. Cytol. 186, 179-224.[Medline]
Hatzfeld, M., Green, K. J. and Sauter, H. (2003). Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains. J. Cell Sci. 1, 1219-1233.
He, W. and Li, S. (2000). Congenital cataracts: gene mapping. Hum. Genet. 106, 1-13.[CrossRef][Medline]
Herrmann, H., Fouquet, B. and Franke, W. W. (1989). Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin. Development 105, 279-298.[Abstract]
Hieda, Y., Tsukita, S. and Tsukita, S. (1989). A new high molecular mass protein showing unique localization in desmosomal plaque. J. Cell Biol. 109, 1511-1518.[Abstract]
Hoffmann, I. and Balling, R. (1995). Cloning and expression analysis of a novel mesodermally expressed cadherin. Dev. Biol. 169, 337-346.[CrossRef][Medline]
Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I. and Haase, H. (2002). The carboxyl-terminal region of AHNAK provides a link between cardiac L-type channels and the actin-based cytoskeleton. FASEB J. 16, 1205-1216.
Hollnagel, A., Grund, C., Franke, W. W. and Arnold, H. H. (2002). The cell adhesion molecule M-cadherin is not essential for muscle development and regeneration. Mol. Cell. Biol. 22, 4760-4770.
Huynh, D. P., Tran, T. M., Nechiporuk, T. and Pulst, S. M. (1996). Expression of neurofibromatosis 2 transcript and gene product during mouse fetal development. Cell Growth Differ. 7, 1551-1561.[Abstract]
Jakobs, P. M., Hess, J. F., FitzGerald, P. G., Kramer, P., Weleber, R. G. and Litt, M. (2000). Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament structural protein gene BFSP2. Am. J. Hum. Genet. 66, 1432-1436.[CrossRef][Medline]
Jensen, O. N., Podtelejnikov, A. and Mann, M. (1996). Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. Rapid Commun. Mass Spectrom. 10, 1371-1378.[CrossRef][Medline]
Karashima, T. and Watt, F. M. (2002). Interaction of periplakin and envoplakin with intermediate filaments. J. Cell Sci. 115, 556-563.
Kibbelaar, M. A., Selten-Versteegen, A.-M. E., Dunia, I., Benedetti, E. L. and Bloemendal, H. (1979). Actin in mammalian lens. Eur. J. Biochem. 95, 543-549.[Abstract]
Koch, P. J., Walsh, M. J., Schmelz, M., Goldschmidt, M. D., Zimbelmann, R. and Franke, W. W. (1990). Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin subfamily of cell adhesion molecules. Eur. J. Cell Biol. 53, 1-12.[Medline]
Krutovskikh, V. and Yamasaki, H. (2000). Connexin gene mutations in human genetic diseases. Mut. Res. 462, 197-207.[CrossRef][Medline]
Langbein, L., Grund, C., Kuhn, C., Praetzel, S., Kartenbeck, J., Brandner, J. M., Moll, I. and Franke, W. W. (2002). Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom. Eur. J. Cell Biol. 81, 419-435.[Medline]
Leckband, D. and Sivasankar, S. (2000). Mechanism of homophilic cadherin adhesion. Curr. Opin. Cell Biol. 12, 587-592.[CrossRef][Medline]
Lee, A., Fischer, R. S. and Fowler, V. M. (2000). Stabilization and remodeling of the membrane skeleton during lens fiber cell differentiation and maturation. Dev. Dyn. 217, 257-270.[CrossRef][Medline]
Leong, L., Menko, A. S. and Grunwald, G. B. (2000). Differential expression of N- and B-cadherin during lens development. Invest. Ophthalmol. Vis. Sci. 41, 3503-3510.
Lieska, N., Chen, J., Maisel, H. and Romero-Herrera, A. E. (1980). Subunit characterization of lens intermediate filaments. Biochem. Biophys. Acta 626, 136-153.[Medline]
Lo, W. K. (1988). Adherens junctions in the ocular lens of various species. Ultrastructural analysis with an improved fixation. Cell Tissue Res. 254, 31-40.[Medline]
Lo, W. K., Shaw, A. P. and Wen, X. J. (1997). Actin filament bundles in cortical fiber cells of the rat lens. Exp. Eye Res. 65, 691-701.[CrossRef][Medline]
Lo, W. K., Shaw, A. P., Paulsen, D. F. and Mills, A. (2000). Spatiotemporal distribution of zonulae adherens and associated actin bundles in both epithelium and fiber cells during chicken lens development. Exp. Eye Res. 71, 45-55.[CrossRef][Medline]
Ma, A. S.-P. and Sun, T.-T. (1986). Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes. J. Cell Biol. 103, 41-48.[Abstract]
Maisel, H., Hardling, C. V., Alcala, J. R., Kuszak, J. and Bradley, R. (1981). The morphology of the lens. In Molecular and Cellular Biology of the Eye Lens (ed. H. Bloemendal), pp. 49-84. New York: John Wiley and Sons.
Martinez-Wittingham, F. J., Sellitto, C., Li, L., Gong, X., Brink, P. R., Mathias, R. T. and White, T. W. (2003). Dominant cataracts result from incongruous mixing of wild-type lens connexins. J. Cell Biol. 161, 969-978.
Masanuga, T., Shimizu, H., Ishiko, A., Fujiwara, T., Hashimoto, T. and Nishikawa, T. (1995). Desmoyokin/AHNAK protein localizes to the non-desmosomal keratinocye cell surface of human epidermis. J. Invest. Dermatol. 10, 941-945.
Melendez-Vasquez, C. V., Rios, J. C., Zanazzi, G., Lambert, S., Bretscher, A. and Salzer, J. L. (2001). Nodes of Ranvier form in association with ezrin-radixin-moesin (ERM)-positive Schwann-cell processes. Proc. Natl. Acad. Sci. USA 98, 1235-1240.
Merdes, A., Brunkener, M., Horstmann, H. and Georgatos, S. D. (1991). Filensin: a new vimentin-binding polymerization competent, and membrane-associated protein of the lens fiber cell. J. Cell Biol. 115, 397-410.[Abstract]
Merdes, A., Gounari, F. and Georgatos, S. D. (1993). The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin. J. Cell Biol. 123, 1507-1516.[Abstract]
Mertens, C., Kuhn, C. and Franke, W. W. (1996). Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque. J. Cell Biol. 135, 1009-1025.[Abstract]
Nagafuchi, A. and Takeichi, M. (1989). Transmembrane control of cadherin-mediated cell adhesion: a 94kD protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. Cell Regul. 1, 37-44.[Medline]
Nelson, W. J., Granger, B. L. and Lazarides, E. (1983). Avian lens spectrin: subunit composition compared with erythrocyte and brain spectrin. J. Cell Biol. 97, 1271-1276.[Abstract]
Nie, Z., Ning, W., Amagai, M. and Hashimoto, T. (2000). C-Terminus of desmoyokin/AHNAK protein is responsible for its translocation between the nucleus and cytoplasm. J. Invest. Dermatol. 114, 1044-1049.
Nielsen, P. A., Baruch, A., Giepmans, B. N. and Kumar, N. M. (2001). Characterization of the association of connexins and ZO-1 in the lens. Cell Commun. Adhes. 8, 213-217.[Medline]
Nishi, O., Nishi, K., Akaishi, T. and Shirasawa, E. (1997). Detection of cell adhesion molecules in lens epithelial cells of human cataracts. Invest. Ophthalmol. Vis. Sci. 38, 579-585.[Abstract]
Ozawa, M., Baribault, H. and Kemler, R. (1989). The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711-1717.[Abstract]
Paffenholz, R., Kuhn, C., Grund, C., Stehr, S. and Franke, W. W. (1999). The arm-repeat protein NPRAP (neurojungin) is a constituent of the plaques of the outer limiting zone in the retina, defining a novel type of adhering junction. Exp. Cell Res. 250, 452-464.[CrossRef][Medline]
Peitsch, W. K., Grund, C., Kuhn, C., Schnölzer, M., Spring, H., Schmelz, M. and Franke, W. W. (1999). Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells. Eur. J. Cell Biol. 78, 767-778.[Medline]
Perng, M. D., Cairns, L., van den Ijssel, P., Prescott, A., Hutcheson, A. M. and Quinlan, R. A. (1999). Intermediate filament interactions can be altered by HSP27 and alpha B-crystallin. J. Cell Sci. 112, 2099-2112.
Rafferty, N. S. (1985). Lens morphology. In The Ocular Lens: Structure, Function and Pathology (ed. H. Maisel), pp. 1-60. New York: Marcel Dekker.
Ramaekers, F. C. S. and Bloemendal, H. (1981). Cytoskeletal and contractile structures in lens cell differentiation. In The Ocular Lens: Structure, Function and Pathology (ed. H. Maisel), pp. 85-136. New York: Marcel Dekker.
Ramaekers, F. C. S., Osborn, M., Schmid, E., Weber, K., Bloemendal, H. and Franke, W. W. (1980). Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type. Exp. Cell Res. 127, 309-327.[Medline]
Repasky, E. A., Granger, B. L. and Lazarides, E. (1982). Widespread occurrence of avian spectrin in nonerythroid cells. Cell 29, 821-833.[Medline]
Rose, O., Grund, C., Reinhardt, S., Starzinski-Powitz, A. and Franke, W. W. (1995). Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus. Proc. Natl. Acad. Sci. USA 92, 6022-6026.
Ruhrberg, C., Hajibagheri, M. A. N., Parry, D. A. D. and Watt, F. M. (1997). Periplakin, a novel component of cornified envelopes and desmosomes that belongs to the plakin family and forms complexes with envoplakin. J. Cell Biol. 139, 1835-1849.
Saika, S., Kawashima, Y., Miyamoto, T., Okada, Y., Tanaka, S., Yamanaka, O., Ohnishi, Y., Ooshima, A. and Yamanaka, A. (1998). Immunolocalization of hyaluronan and CD44 in quiescent and proliferating human lens cells. J. Cataract Refract. Surg. 24, 1266-1270.[Medline]
Schmelz, M. and Franke, W. W. (1993). Complexus adhaerentes, a new group of desmoplakin-containing junctions in endothelial cells: the syndesmos connecting retothelial cells of lymph nodes. Eur. J. Cell Biol. 61, 274-289.[Medline]
Schmidt, A., Heid, H. W., Schäfer, S., Nuber, U. A., Zimbelmann, R. and Franke, W. W. (1994). Desmosomal and cytoskeletal architecture in epithelial differentiation: cell-type specific plaque components and intermediate filament anchorage. Eur. J. Cell Biol. 65, 229-245.[Medline]
Schmidt, A., Langbein, L., Prätzel, S., Rode, M., Rackwitz, H.-R. and Franke, W. W. (1999). Plakophilin-3 a novel cell-type specific desmosomal plaque protein. Differentiation 64, 291-306.[CrossRef][Medline]
Schröder, R., Warlo, I., Herrmann, H., van der Ven, P. F. M., Klasen, C., Blümcke, I., Mundegar, R. R., Fürst, D. O., Goebel, H. H. and Magin, T. M. (1999). Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle. Eur. J. Cell Biol. 78, 288-295.[Medline]
Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. S., Grübel, G., Legrand, J.-F., Als-Nielsen, J., Colman, D. R. and Hendrickson, W. A. (1995). Structural basis of cell-cell adhesion by cadherins. Nature 374, 327-337.[CrossRef][Medline]
Sherman, D. L. and Brophy, P. J. (1999). A tripartite nuclear localization signal in the PDZ-domain protein L-periaxin. J. Biol. Chem. 275, 4537-4540.
Shtivelman, E. and Bishop, J. M. (1993). The human gene AHNAK encodes a large phosphoprotein located primarily in the nucleus. J. Cell Biol. 120, 625-630.[Abstract]
Shtivelman, E., Cohen, F. E. and Bishop, J. M. (1992). A human gene (AHNAK) encoding an unusually large protein with a 1.2-µm polyionic rod structure. Proc. Natl. Acad. Sci. USA 89, 5472-5476.[Abstract]
Simon, M. and Green, H. (1984). Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36, 827-834.[Medline]
Simonneau, L. and Thiery, J. P. (1998). The mesenchymal cadherin-11 is expressed in restricted sites during the ontogeny of the rat brain in modes suggesting novel functions. Cell Adhes. Commun. 6, 431-450.[Medline]
Simonneau, L., Kitagawa, M., Suzuki, S. and Thiery, J. P. (1995). Cadherin-11 expression marks the mesenchymal phenotype: towards new functions for cadherins? Cell Adhes. Commun. 3, 115-130.[Medline]
Staehelin, L. A. (1974). Structure and function of intercellular junctions. Int. Rev. Cytol. 39, 191-283.[Medline]
Sussmann, J., Stokoe, D., Ossina, N. and Shtivelman, E. (2001). Protein kinase B phosphorylates AHNAK and regulates its subcellular localization. J. Cell Biol. 154, 1019-1030.
Takai, Y. and Nakanishi, H. (2003). Nectin and afadin: novel organizers of intercellular junctions. J. Cell Sci. 116, 17-27.
Takeichi, M. (1988). The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development 102, 639-655.[Abstract]
Takeichi, M. (1991). Cadherin adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455.[Medline]
Thomas, G. H. (2001). Spectrin: the ghost in the machine. BioEssays 81, 152-160.[CrossRef]
Toyofuku, T., Yabuki, M., Otsu, K., Kuzuya, T., Hori, M. and Tada, M. (1998). Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes. J. Biol. Chem. 273, 12725-12731.
Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A. and Tsukita, S. (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J. Cell Biol. 126, 391-401.[Abstract]
Valiron, O., Chevrier, V., Usson, Y., Breviario, F., Job, D. and Dejana, E. (1996). Desmoplakin expression and organization at human umbilical vein endothelial cell-to-cell junctions. J. Cell Sci. 109, 2141-2149.
Volk, T. and Geiger, B. (1984). A 135-kD membrane protein of intercellular adherens junctions. EMBO J. 3, 2249-2260.[Abstract]
Volk, T., Cohen, O. and Geiger, B. (1987). Formation of heterotypic adherens-type junctions between L-CAM-containing liver cells and A-CAM-containing lens cells. Cell 5, 987-994.
Weitzer, G. and Wiche, G. (1987). Plectin from bovine lenses. Chemical properties, structural analysis and initial identification of interaction partners. Eur. J. Biochem. 169, 41-52.[Abstract]
Yonemura, S., Hirao, M., Doi, Y., Takanahashi, N., Kondo, T., Tsukita, S. and Tsukita, S. (1998). Ezrin/Radixin/Moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43 and ICAM-2. J. Cell Biol. 140, 885-895.