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A protein recently described as a desmosome-specific molecule involved in anchoring intermediate-sized filaments (IFs) to the desmosomal plaque, and hence named "pinin" [43], has been known in our laboratory for several years as a strictly nuclear protein occurring in a wide range of cell types, including many that are totally devoid of desmosomes. Using a series of specific antibodies we have localized the protein in the nucleoplasm of cultured cells, blood cells and solid tissues of diverse vertebrate species, from oocytes to erythrocytes of amphibia and from liver to connective tissue and fibroblasts in mammals. Desmosomes have consistently been negative, and the nuclear specificity of the immunolocalization reactions has also been directly demonstrated by double-label immunofluorescence microscopy. From our results we conclude that this nuclear protein, characterized by a domain exceptionally rich in serine residues and hence termed DRS-protein, occurs in at least two genetically different forms in a diffusible state as well as in special ribonucleoprotein-particles, "speckles" [6], and is a widespread if not ubiquitous nuclear protein. Consequently it must serve nuclear functions rather than "pinning" IFs to plasma membranes and does not provide a new reliable marker for desmosomes and epithelial or myocardial differentiation.
Fig. 1a–a′ Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE; 10% gel) and immunological identification
of the “domain rich in serine†(DRS) polypeptide in Xenopus laevis
kidney epithelial (XLKE, line A6) cells and in mass-isolated
nuclei of Xenopus laevis oocytes. a Coomassie brilliant blue staining
of reference proteins (lane 1; from top to bottom 205 kDa, 116
kDa, 97 kDa, 66 kDa, 45 kDa, 29 kDa), and of total proteins from
XLKE cells (lane 2) and from mass-isolated oocyte nuclei (lane
3). a′, a′ Enhanced immuno-chemiluminescence (ECL) detection
of DRSP (arrowheads) at a position corresponding to approx. 160
kDa by two different guinea pig antibody preparations against
peptides deduced from Xenopus laevis cDNA clone DRSP-MX11
(a′ antibodies DRSP-B2, a′ antibodies DRSP-B4). The lower
band positive in a lane 3′ has not been identified yet, but is probably
a proteolytic breakdown product. The polypeptide synthesized
in vitro from cDNA DRSP-MX3 by coupled in vitro transcription/
translation migrates exactly at the position of the bands denoted
here by arrowheads (see also [6])..:
Fig. 2 Two-dimensional gel electrophoresis of DRSP separated by
non-equilibrium pH-gradient electrophoresis (NEPHGE; horizontal
arrows) in the first and SDS-PAGE (downward arrows) in the second
dimension; upper panel Ponceau S-stained polypeptides of the
remaining material of Xenopus laevis kidney epithelial cells
(XLKE) of line A6 extracted with 0.5% Triton and 0.1 M NaCl; A
α-actin, B BSA, PGK phosphoglycerolkinase, R reference proteins
(see Fig. 1); lower panel corresponding immunoblot reaction, visualized
by ECL, shows the position of the DRS-protein at a pI of approximately
5.4 and a Mr value of about 160000 (arrow). Most of
the protein, however, has remained at the start position of the firstdimension
gel electrophoresis (indicated by an asterisk).:cgi&/
Fig. 3a–a′ Immunofluorescence microscopy of DRSP in Xenopus
laevis cultured kidney epithelial cells (XLKE, line A6) grown on
coverslips, (a, epifluorescence; a′ corresponding phase contrast).
DRSP is restricted to nuclei where it is distributed in a finely granular
fashion throughout the nucleoplasm, excluding the nucleoli.
During mitosis the protein is dispersed throughout the cytoplasm,
excluding the mitotic chromosomes (see cell in the central bottom
part). Desmosomes present in these cells are negative. Bar 20 μm.:
Fig. 4a–b′ Immunofluorescence microscopy on frozen sections
through Xenopus laevis tissues, demonstrating the nuclear localization
of DRSP in all cell types present. a, a′ Skin, including epidermal
keratinocytes (a) epithelial cells of a glandular duct (G
central cell column in a′), and the non-epithelial cells of the connective
tissue (CT; a is epifluorescence, a′ is a phase-contrast image.
Some positive nuclei are denoted by arrows). b, b′ Small intestine
(L lumen, S submucosa; epithelial cell layer is denoted by
brackets in b′; b is epifluorescence and b′ phase contrast; Note,
besides the numerous epithelial nuclei, a positive reaction also in
the nuclei of submucosal cells). Bars, 20 μm.:
