Download visualization of charged groups on the surface of rat liver nuclei

J. Cell Sci. 16, 665-675 (i974)
Printed in Great Britain
665
VISUALIZATION OF CHARGED GROUPS ON
THE SURFACE OF RAT LIVER NUCLEI
ISMO VIRTANEN* AND JORMA WARTIOVAARA
Third Department of Pathology and the Electron Microscope
Laboratory, University of Helsinki, Helsinki, Finland
SUMMARY
Anionic groups on the outer surfaces of isolated rat liver nuclei were rendered visible in
the electron microscope by staining with colloidal iron hydroxide at different pH values. At
pH i-8 the nuclei did not adsorb particles of stain, although plasma membranes left in the same
preparation showed heavy labelling. After pretreatment with neuraminidase at pH 6 the plasma
membranes were no longer stained. At pH 3-0 the nuclear surfaces also stained intensely. The
staining pattern acquired at this pH did not appear to be changed by neuraminidase pretreatment.
With the staining method used, rat liver nuclear surfaces seemed to have no exposed sialic
acid under isolation conditions which preserve the nuclear membranes and leave the ribosomes
attached to the nuclear surface. However, at higher pH values other anionic groups seem to
become dissociated and are stained with colloidal iron hydroxide.
INTRODUCTION
Cells have been shown to carry various anionic groups on their surface membranes,
as demonstrated by biochemical (Winzler, 1970), cell-electrophoretical (Doljanski &
Eisenberg, 1965; Weiss, 1969), and ultrastructural methods (Benedetti & Emmelot,
1967; Weiss & Zeigel, 1972). It has been suggested that sialic acid, ribonucleic acid
(Weiss, 1969) and in some cells sulphated groups (Marx, Graf & Wesemann, 1973)
contribute to the negative surface charge observed on the surfaces of these cells.
The presence on plasma membranes of macromolecules containing sialic acid,
glycoprotcins or glycolipids has been studied with particular interest, as these molecules have been postulated to contribute to various cellular receptors, and to recognition and adhesion processes (Gielen, 1968; Emmelot, 1973; Weiss, 1973).
Related biological roles have also been suggested for glycoproteins containing sialic
acid on internal cellular membranes (Bosmann, 1973). However, whether sialic
acid and other saccharides are present on the cytoplasmic surface of internal cellular
membranes is still controversial (Hirano et al. 1972). As regards rat liver nuclear
surfaces, the presence of charged groups and sialic acid has been studied with both
cell-electrophoretical (e.g. Bosmann, 1973) and biochemical (e.g. Zbarsky, 1972)
methods using isolated nuclei. These studies have yielded rather mixed results,
probably depending partly on differences in isolation methods and experimental
* Address for correspondence: Ismo Virtanen, Third Department of Pathology, University
of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland.
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/. Virtanen andj. Wartiovaara
conditions. To provide ultrastructural confirmation of these studies, we undertook
to investigate the presence of charged groups on isolated rat liver nuclei with an
electron-microscope marker, colloidal iron hydroxide (CIH), known to be rather
specific for groups containing sialic acid at pH values 1*7—1-8 (Benedetti & Emmelot,
1967; Weiss, Zeigel, Jung & Bross, 1972; Nicolson, 1973).
MATERIALS AND METHODS
Nuclear isolation
For each experiment 2-4 rats were decapitated and their livers rapidly removed and suspended in cold physiological saline. The livers were minced with scissors in ice-cold 032 M
sucrose solution containing 3 mM MgCl 2 . Homogenization was performed in 5 volumes of the
same solution with a Teflon pestle homogenizer at low speed. The diluted homogenate (2 x )
was filtered through 2 layers of cheesecloth and centrifuged at 1000 rev/min for 10 min in an
IEE model PR6 centrifuge. After centrifugation the supernatant was decanted and the pellet
resuspended with a Vortex mixer in a 0 8 8 M sucrose solution containing 2 mM MgCL. Final
purification of the nuclei was carried out with sucrose gradient ultracentrifugation. The samples
were pipetted on the top of sucrose step gradient layers of 1-5, 1 8 and 2-3 M in 2 mM MgCL.
In some gradients the lowermost layer was replaced with 2 M sucrose solution to increase the
yield of plasma membranes. Ultracentrifugation was performed in a Spinco Model 50L
centrifuge (Beckman Inc.) with a SW 25. i L rotor for 60 min at 22000 rev/min. The nuclear
pellets obtained were washed several times in 025 M sucrose-3 mM MgCl 2 solution and processed for enzyme treatments or electron microscopy.
Enzyme treatment
For enzyme digestions isolated nuclei were incubated for 30 min at 30 °C in a solution containing 50 U./ml (at 37 °C) neuraminidase (Behringwerke AG, Vibrio comma) in C25 M
sucrose-2 mM CaCL buffered with 0 0 1 M Tris-maleate to p H 6-0 (Drzeniek, 1973). After the
treatment the samples were immersed in an ice bath and pelleted twice for 10 min at 1000
rev/min in an IEE model PR6 centrifuge at 4 °C in 0-25 M sucrose-3 m M MgCl 2 solution
before processing for electron microscopy.
Electron microscopy
For electron microscopy resuspended nuclei were fixed for 30 min in ice-cold 2-5 %
glutaraldehyde buffered with 0 1 M sodium cacodylate to p H 7-2. Colloidal iron hydroxide
(CIH) staining (Benedetti & Emmelot, 1967) was performed with carefully resuspended fixed
nuclei in order to ensure good exposure to stain particles. The p H of the staining solution was
adjusted to p H 1-8 with acetic acid and to pH 3-0 with NaOH. After exposure to the stain for
1 h at room temperature the samples were pelleted, fixed in 1-5 % osmium tctroxide and embedded in Epon 812. Thin sections were post-stained with uranyl acetate and lead citrate or
left unstained for easier visualization of CIH stain particles. Philips EM300 or Jeol 100B
electron microscopes were used at an accelerating voltage of 80 kV.
RESULTS
Our method for isolation of rat liver nuclei was essentially a modification of the
method developed by Incefy & Kappas (1971) for isolation of nuclei from chick
embryo liver. The conditions of the isolation media, including the omission of buffers,
were so chosen as to preserve the general ultrastructure of the nuclei and the integrity
of the nuclear membranes and to retain the membrane-attached ribosomes on the
outer nuclear membrane (Incefy & Kappas, 1971; Laval & Bouteillc, 1973). In some
Charged groups on nuclei
667
experiments the yield of identifiable plasma membrane fragments was increased by
modifying the gradients, to obtain an internal control for our staining method.
The isolated nuclei had a well-preserved ultrastructure (Fig. 1). Their chromatin
was unaggregated and the nuclear membranes were mostly intact. Ribosomes were
found in varying amounts on the outer nuclear membranes (Fig. 2 A, B).
After the fixed preparations had been stained with colloidal iron hydroxide (CIH)
solution at pH i-8, the plasma membranes left in the preparation showed dense
labelling with stain particles (Figs. 3, 4). However, the nuclear surfaces seen in the
same sections had only a few stain particles attached (Figs. 3, 5).
Neuraminidase treatment of the specimens (50 U./ml, 30 min at 30 °C, pH 6-o)
removed most of the stain from the plasma membranes (Fig. 6) but had no apparent
influence on the staining properties of the nuclear surfaces.
When stained at pH 3-0, both the plasma membranes and the outer nuclear
membranes of the isolated nuclei carried heavy deposits of stain particles (Figs. 7, 8).
The heavy CIH-staining pattern at pH 3-0 was not apparently altered by pretreatment
of the plasma membranes and nuclei with neuraminidase (Figs. 9, 10).
DISCUSSION
Colloidal iron hydroxide staining (CIH) has been shown to be a relatively specific
staining method for surface-bound iV-acetyl neuraminic acid (a sialic acid) at pH
values 1-7 to i-8 (Benedetti & Emmelot, 1967; Weiss et al. 1972; Nicolson, 1973).
At this pH only a few groups with low pK values can be ionized. ./V-acetyl neuraminic
acid, with a pK value of 27, is partially charged (Drzeniek, 1973). In addition, it has
been suggested that sulphate groups with a pK value of 1-9 (Marx et al. 1973) and
the first phosphate groups of surface-bound ribonucleic acid, pK i-o (Weiss & Zeigel,
1972) may also contribute to the staining of membranes with CIH at pH i-8. However,
whether these 2 latter groups are present on cellular membranes is uncertain
(Emmelot, 1973).
In our study the outer surfaces of rat liver cell plasma membranes showed dense
labelling with CIH at pH i-8, as also reported earlier by Benedetti & Emmelot (1967).
However, no stain could be seen on the surfaces of the isolated rat liver nuclei. The
lack of stain on the nuclei cannot have been due to penetration artifacts during the
staining process, because the samples were thoroughly dispersed in the staining
solution. In addition, in cases of appositionally situated nuclei and plasma membranes
only the nuclear surfaces lacked the stain. The pK value of the first phosphate group
of RNA is i-o (Weiss & Zeigel, 1972) and it has been suggested that ribosomal RNA
is largely exposed on the ribosomal surface (Cox & Bonanou, 1969). However, at pH
i*8 we did not obtain any staining of ribosomes attached to the nuclear surface.
The disappearance of the staining of rat-liver-cell plasma membranes on treatment
with neuraminidase supports the view that at pH i-8 the attachment of CIH particles
depends solely on the presence of carboxyl groups contributed by sialic acid residues.
In this connexion it is interesting to note that raising the pH of the CIH staining
solution to pH 3-0 causes new stainable neuraminidase-resistant groups to emerge
both on the plasma membrane and on the nuclear surface. These groups may
668
/. Virtanen andj. Wartiovaara
correspond to the carboxyl groups of amino acids, with a pK range of 3 to 4-0, which
have been shown by cell electrophoresis to be exposed on cell surfaces at least (Vassar
& Kendall, 1969). In earlier studies carboxyl groups contributed by sialic acid residues have been detected biochemically in the nuclei of rat liver cells (Kawasaki &
Yamashina, 1972; Zbarsky, 1972; Phillips, 1973), L cells (Glick, Comstock, Cohen &
Warren, 1971) and BHK cells (Keshgegian & Glick, 1973). On the other hand,
Kashnig & Kasper (1969) reported only a negligible amount of sialic acid associated
with rat liver cell nuclei. It seems that the conflicting results depend on the different
techniques used in the isolation of nuclei and measurement of sialic acid (Keshgegian
& Glick, 1973). At present it cannot be decided whether sialic acids are present on
the surface of the outer nuclear membrane or on the cisternal surface of the nuclear
envelope.
With cell electrophoresis, somewhat varying results have been obtained concerning
the presence of sialic acid on rat liver nuclear surfaces (Kishimoto & Liebermann,
1964; Mayhew & Nordling, 1966; Vassar, Seaman, Dunn & Kanke, 1967; Bosmann,
1973). In a recent study Bosmann (1973) reported a marked decrease in mobility
after neuraminidase treatment of rat liver cell nuclei isolated by the sucrose method.
Although this study lacked ultrastructural demonstration that the nuclei were intact,
the author suggested that sialic acid was present on the nuclear surface. However,
in one fundamental respect the experimental conditions used by Bosmann differed
from those used here: the solutions he used in electrophoresis lacked divalent cations.
This probably caused detachment of the ribosomes bound to the nuclear envelope,
which are known to require divalent cations for their attachment to membranes
(Sabatini et al. 1972). Therefore, our observation that groups containing sialic acid
are not exposed on the nuclear surfaces does not conflict with Bosmann's electrophoretic results, if one assumes that these groups were covered by ribosomes preserved
on the nuclear surfaces under our isolation conditions, including magnesiumcontaining media. According to Scott-Burden & Hawtrey (1973), the attachment of
ribosomes to rat liver ER membranes is sensitive to neuraminidase treatment. This
seems to indicate that sialic acid participates in ribosome binding to membranes.
Whether under our conditions neuraminidase causes detachment of ribosomes from
isolated rat liver nuclei is now being investigated.
This study was supported by grants from the Damon Runyon Memorial Foundation, the
Sigrid Juselius Foundation and the National Research Council for Medical Sciences, Finland.
REFERENCES
BENEDETTI, E. L. & EMMELOT, P. (1967). Studies on plasma membranes. IV. The ultrastructural
localization and content of sialic acid in plasma membranes isolated from rat liver and
hepatoma. J. Cell Sci. 2, 499-512.
BOSMANN, H. B. (1973). Molecules at the external nuclear surface. Sialic acid of nuclear
membranes and electrophoretic mobility of isolated nuclei and nucleoli. J. Cell Biol. 59,
601-614.
Cox, R. A. & BONANOU, S. A. (1969). A possible structure of the rabbit reticulocytc ribosome.
Biochem.J. 114, 769-774.
Charged groups on nuclei
669
DOLJANSKI, F. & EISENBERG, S. (1965). The action of neuraminidase on the electrophoretic
mobility of liver cells. In Cell Electrophoresis (ed. E. J. Ambrose), pp. 78-84. London:
Churchill.
DRZENIEK, R. (1973). Substrate specificity of neuraminidases. Histochem.J. 5, 271-290.
EMMELOT, P. (1973). Biochemical properties of normal and neoplastic cell surfaces: a review.
Enr.J. Cancer 9, 319-333.
GIELEN, W. (1968). Vorkommen und biologische Bedeutung der Neuraminsaure. Naturwissenschaften 55, 104—109.
GLICK, M. C., COMSTOCK, C. A., COHEN, M. A. & WARREN, L. (1971). Membranes of animal
cells. VIII. Distribution of sialic acid, hexosamines and sialidase in the L cell. Biochim.
biophys. Ada 233, 247-257.
HIRANO, H., PARKHOUSE, P., NICOLSON, G. L., LENNOX, E. S. & SINGER, S. J. (1972). Distri-
bution of saccharidc residues on membrane fragments from a myeloma-cell homogenate:
its implication for membrane biogenesis. Proc. natn. Acad. Sci. U.S.A. 69, 2945-2949.
INCEFY, G. S. & KAPPAS, A. (1971). Isolation and biochemical characterization of nuclei from
chick embryo liver. J. Cell Biol. 50, 385-398.
KASHNIG, D. M. & KASPER, C. B. (1969). Isolation, morphology, and composition of the nuclear
membrane from rat liver. J. biol. Chem. 244, 3786-3792.
KAWASAKI, T. & YAMASHINA, I. (1972). Isolation and characterization of glycopeptides from
rat liver nuclear membrane. J. Biochem., Tokyo 72, 1517-1525.
KESHGEGIAN, A. A. & GLICK, M. C. (1973). Glycoproteins associated with nuclei of cells
before and after transformation by a ribonucleic acid virus. Biochemistry, N. Y. 12, 1221-1226.
KISHIMOTO, S. & LIEBERMAN, I. (1964). A nuclear membrane change after partial hepatectomy.
y. Cell Biol. 23, s 11-518.
LAVAL, M. & BOUTEILLE, M. (1973). Synthetic activity of isolated rat liver nuclei. I. Ultrastructural study at various steps of isolation. Expl Cell Res. 76, 337-348.
MARX, R., GRAF, E. & WESEMANN, W. (1973). Histochemical and biochemical demonstration
of sialic acid and sulphate in vesicles and membranes isolated from nerve endings of rat
brain. J. Cell Sci. 13, 237-255.
MAYHEW, E. & NORDLING, S. (1966). Electrophoretic mobility of mouse cells and homologous
isolated nuclei. J. cell. Physiol. 68, 75-80.
NICOLSON, G. L. (1973). Cis- and trans-membrane control of cell surface topography.
J. supramolec. Structure 1, 410-416.
PHILLIPS, J. L. (1973). Carbohydrate composition of rat hepatocyte nuclear membrane as
compared to normal, Morris hepatome 7800, and phenobarbital-induced microsomal
membranes. Arclis Biochem. Biophys. 156, 377-379.
SABATINI, D., BORGESE, N., ADELMAN, M., KREIBICH, G. & BLOBEL, G. (1972). Studies on the
membrane associated protein synthesis apparatus on eukaryotic cells. In RNA Vinisesj
Ribosomes, Proc. 8th Meeting Eur. biochem. Socs (ed. H. Bloemendal, E. M. J. Jaspors,
A. von Krammer & R. S. Planta), pp. 147-171. Amsterdam: North-Holland Publishing.
SCOTT-BURDEN, T. & HAWTREY, A. O. (1973). The effect of neuraminidase treatment of
ribosome-frec membranes on their ribosomal reattachment ability. Biochem. biophys. Res.
Commurt. 54, 1288-1295.
VASSAR, P. S., SEAMAN, G. V. F., DUNN, W. L. & KANKE, L. (1967). Electrokinetic properties
of nuclear surfaces. A comparison of nuclei from normal and regenerating rat liver. Biochim.
biophys. Acta 135, 218-224.
VASSAR, P. S. & KENDALL, M. J. (1969). Electrophoresis of human leukocytes. Archs Biochem.
Biophys. 135, 35O-35SWEISS, L. (1969). The cell periphery. Int. Rev. Cytol. 26, 63-105.
WEISS, L. (1973). Neuraminidase, sialic acids, and cell interactions.^, natn. Cancer Inst. 50,
3-i9WEISS, L. & ZEIGEL, R. (1972). Cell surface negativity and the binding of positively charged
particles. J . cell. Physiol. 77, 179-186.
WEISS, L., ZEIGEL, R., JUNG, O. S. & BROSS, I. D. J. (1972). Binding of positively charged
particles to glutaraldehyde-fixcd human erythrocytes. Expl Cell Res. 70, 57-64.
WINZLER, R. J. (1970). Carbohydrates in cell surfaces. Int. Rev. Cytol. 29, 77-125.
ZBARSKY, I. B. (1972). Nuclear envelope isolation. In Methods in Cell Physiology (ed. D. M.
Prescott), pp. 167-198. New York and London: Academic Press.
{Received 23 April 1974)
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Fig. 1. Electron micrograph of the nuclear fraction obtained by homogenization of
rat liver cells in 0-25 M sucrose-3 mM MgCl2 solution and centrifugation through a
sucrose step gradient. The nuclear chromatin is homogeneously dispersed, and well
developed nucleoli («) are seen. The nuclear membranes (arrows) are well preserved. Uranyl acetate and lead citrate post-staining, x 12000.
Fig. 2A, B. The isolated nuclei carry variable amounts of ribosomes (r) on their
outer membranes. Uranyl acetate and lead citrate post-staining. A and B x 95 0000
and x 73000, respectively.
Charged groups on nuclei
2B
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/. Virtanen andj. Wartiovaara
Fig. 3. Appositionally located rat liver-cell plasma membrane (/>»«) and nuclear
membrane (nm) stained with colloidal iron hydroxide (CIH) at pH i-8 but without
post-staining. Numerous electron-dense CIH granules are attached to the plasma
membrane but are almost totally lacking from the nuclear membrane, x 60000.
Fig. 4. At higher magnification CIH particles are seen to be densely deposited on the
plasma membrane stained at pH i'8. In an area where the surface membrane has
apparently been tangentially sectioned (arrow) the CIH particles are randomly dispersed on the surface of the membrane, x 120000.
Fig. 5. Closer view of an isolated nucleus stained with CIH at pH 1 -8. The ribosomes
(r) bound to the outer nuclear membrane seem to lack the stain particles. A few
particles are associated with a nuclear pore-like structure (arrow) and are also seen
in the nucleoplasm. x 120000.
Fig. 6. After neuraminidase preincubation the plasma membrane (pm) shows greatly
reduced CIH staining at pH i-8. x 75000.
Charged groups on nuclei
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I- Virtanen andjf. Wartiovaara
Fig. 7. Plasma membrane stained with CIH at pH 3-0. The staining is dense in both
transversely and tangentially (arrow) sectioned parts of the membrane, x 75000.
Fig. 8. After staining with CIH at pH 3-0 the nuclear membrane (nm) also carries
dense deposits of stain particles, x 75000.
Fig. 9. Neuraminidase pretreatment does not apparently change the heavy CIH
staining of plasma membranes (pm) at pH 3-0. x 75000.
Fig. 10. After pretreatment with neuraminidase, the nuclear membrane (nm) still
carries dense deposits of CIH particles at pH 3-0. x 75000.
Charged groups on nuclei
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