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The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering
JOURNAL OF COLLOID AND INTERFACE SCIENCE
177, 9–13 (1996)
Article No. 0002
The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering ADELINA PRADO,† CARMEN PUYO,* JON ARLUCEA,* FELIX M. GON˜I,† ,1
AND
JUAN ARECHAGA *
Departments of *Cell Biology and †Biochemistry, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain Received September 27, 1994; accepted May 22, 1995
The movement of water and solutes across membranes in closed vesicles is most conveniently measured following changes in suspension turbidity (1, 2). However, the complex structure of isolated nuclei makes it difficult to relate turbidity changes to changes in volume, in turn attributable to flow of water and/or solutes. Several reports have described turbidity changes due to cation-dependent chromatin condensation (3–5), while the effects of osmotic changes on turbidity and nuclear volume have not been systematically studied, to our knowledge. In order to clarify, at least partially, the origin of changes in the turbidity of nuclear suspensions, we have studied the effect of various ionic and nonionic solutes on isolated nuclei and artificial phospholipid vesicles (liposomes), combining turbidimetric measurements with light-microscopy estimations of nuclear size. We conclude that, apart from chromatin condensation, changes in nuclear size and changes in bilayer structure or phospholipid conformation may also contribute to the observed changes in turbidity of the nuclear suspensions.
The turbidity of a suspension of cell nuclei isolated from animal tissue homogenates is a complex case of non-Rayleigh scattering. As a first approximation to this system, we have characterized a number of factors that may contribute to the observed turbidity: cation-dependent chromatin condensation, thermal denaturation of chromatin, nuclear shrinking, and changes in the optical properties of the membrane bilayer. Small differences in cation concentration, particularly in the case of divalent cations, lead to large changes in chromatin supramolecular organization, thus to large turbidity effects; thermally-induced changes in turbidity have a similar origin, although they are less pronounced. Under certain circumstances, either salts or heat may induce condensation of chromatin, the latter being connected to the inner side of the nuclear envelope, nuclear shrinking ensues, and this in turn modifies the suspension turbidity. Finally, changes in the physical properties of the lipid bilayers or of the phospholipids in the nuclear envelopes may also have significant effects, though smaller than chromatin changes, in the overall turbidity of the nuclear suspension. q 1996 Academic Press, Inc. Key Words: nucleus, cell; organelles, cell; chromatin; bilayers, lipid; light scattering.
MATERIALS AND METHODS INTRODUCTION
Mouse liver nuclei were isolated as in (6); purity was checked by microscopic, chemical, and enzymatic methods and found to be at least comparable to that of other preparations (7, 8). Turbidity was measured at room temperature, as the apparent absorbance at 540 nm, in a PW 8625 UV/ Vis Philips spectrophotometer. This is a commonly used wavelength for measuring the turbidity of subcellular organelle suspensions (9); it is low enough to produce a convenient intensity of scattering and biomolecules do not absorb strongly at this wavelength. In our case, a linear relation was observed between 540 nm scattered light and nuclear concentration, in the range 0.20–0.85 absorbance units. Protein concentration in the cuvette was 0.7 mg/ml. Nuclear sizes were evaluated by light microscopy, using a Leitz Labovert inverted microscope. The unstained nuclei were observed by the phase-contrast technique, with a magnification of 401. Nuclear diameters were measured with a standard grating ruled at normal angle and calibrated with latex micro-
Light scattering by large, heterogeneous particles presents considerable theoretical and practical problems for its analysis. Yet common use is made of light scattering or ‘‘turbidity’’ measurements in biological studies of subcellular organelles and even whole cells in suspension, particles unavoidably large and heterogeneous from the point of view of Rayleigh and related approximations. One such case is presented by the suspensions of isolated nuclei from plant or animal cells: nuclei are approximately spherical vesicles, with a diameter in the 1-mm range, limited by two membranes (each consisting of a lipid bilayer), and containing an aqueous dispersion of chromatin, a mixture of nucleic acids and proteins. The present work constitutes a first approximation to the analysis of the various factors involved in the turbidity of nuclear suspensions. 1
To whom correspondence should be addressed. 9
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0021-9797/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PRADO ET AL.
spheres (4.2, 6.44, and 9.6 mm) (Polysciences, Inc. Polybead–Polystyrene Microspheres). For each data point 100 nuclei were measured. Unless otherwise stated, nuclei were resuspended in 5 mM Tris–HCl, pH 7.4, 1.73 mM sucrose, 0.6 mM KCl, 0.06 mM MgCl2 (standard buffer). For thermal treatments nuclei were incubated for 15 min at the desired temperature, then left to equilibrate at room temperature before the measurements. Osmolalities were measured in a Osmomat 030 (Gonotec). Polystyrene microspheres were from Polysciences, Inc. Multilamellar vesicles (liposomes) composed of eggyolk phosphatidylcholine and dicetylphosphate (10:1 mol ratio) were prepared and osmotically tested as in (1, 2). Chromatin was isolated from mouse liver nuclei as described in (4). Briefly, a sediment of isolated nuclei was resuspended to a concentration of 5 1 10 8 nuclei/ml in 10 mM triethanolamine/HCl, 10% (w/v) sucrose, 0.1 mM MgCl2 , pH 7.4 buffer and incubated with DNase I (25 mg/ml) at 377C for 15 min. The digestion was stopped by adding a threefold excess of ‘‘stop buffer’’ (10 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl2 , 0.5 mM PMSF, pH 7.5) at 47C. The suspension was centrifuged at 5000 1 g, 47C, 10 min, and the clear supernatant was discarded. The resulting sediment was then resuspended in four volumes of ‘‘lysis buffer’’ (10 mM Tris–HCl, 1 mM Na3EDTA, 0.1 mM PMSF, pH 7.5) and centrifuged at 20000 1 g, 47C, 10 min. The supernatant, containing the chromatin, was diluted until an A540 of É0.35 was obtained in the presence of 10 mM MgCl2 . RESULTS
Among the various cations that are known to produce changes in the turbidity of nuclei in suspension, magnesium was selected as the main divalent cation in our study because of its known affinity for the phosphate groups of nucleotides and nucleic acids. The effect of magnesium ions on the turbidity of isolated mouse liver nuclei in suspension is shown in Fig. 1A. Magnesium, in the 10 04 –10 02 M concentration range, increases the suspension turbidity. Light-microscopy observations ensure that nuclei do not aggregate in the presence of Mg 2/ in our preparations (data not shown). This experiment was first performed by adding increasing concentrations of MgCl2 , i.e., varying simultaneously Mg 2/ concentration and osmotic pressure. Both factors have then been separately studied, by repeating the experiment at increasing or constant (sucrose-compensated) osmotic pressure (Fig. 1). Changes in turbidity (Fig. 1A) are virtually the same either at constant or variable osmotic pressure. Examining the data in the light of osmolality measurements (Figs. 1A and 1C) it is seen that a large increase in turbidity occurs between 10 04 and 2 1 10 03 M Mg 2/ , while the corresponding increase in osmolality is negligible, thus confirming the idea that changes in turbidity are unrelated to changes in osmotic pressure. However, the data
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FIG. 1. Effect of magnesium ions on nuclear suspension turbidity and nuclear size. Nuclei in standard buffer (see Methods) were transferred into different MgCl2 solutions and incubated for 5 min at room temperature prior to the measurements. Experiments were carried out keeping constant the osmolality of the MgCl2 solutions, by substituting sucrose for MgCl2 (filled symbols) or under conditions of varying osmolality (open symbols).
in Fig. 1B reveal that the observed increase in turbidity is concomitant with a decrease in nuclear size; nuclear shrinking may certainly be responsible, either fully or in part, for the said increase in turbidity.
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TURBIDITY OF CELL NUCLEI IN SUSPENSION
Above 7 1 10 02 M Mg 2/ , nuclear turbidity falls precipitously. Microscopic controls of the morphology of nuclei in suspension failed to reveal any significant nuclear disruption at these high Mg 2/ concentrations that could give rise to a decrease in turbidity. Other divalent cations (Ca 2/ , Mn 2/ ) had exactly the same effects as Mg 2/ , at similar concentrations, and unrelated to osmotic pressure. Monovalent cations (Li / , Na / , K / , Cs / ) and Tris also produced an increase in turbidity, but at higher concentrations ( ú10 02 M), that was followed by an abrupt decrease beyond 0.2 M (data not shown). No differences between individual monovalent cations were observed. In an effort to dissect the various sources of nuclear suspension turbidity, the effect of increasing Mg 2/ concentrations was separately tested on two different systems; these may be considered to represent the two main nuclear components from the point of view of turbidity, namely isolated chromatin (Fig. 2A) and chromatin-free phospholipid vesicles (Fig. 2B), the latter mimicking the nuclear envelopes. The effect of Mg 2/ on isolated chromatin reproduces the main features of its effect on isolated nuclei, particularly the increase and, later, abrupt decrease in turbidity (Figs. 1A and 2A). Note that chromatin concentration is the same in both experiments. However, for free chromatin turbidity increases significantly only above 2 1 10 03 M Mg 2/ , while a comparable effect is caused on intact nuclei by cation concentrations one order of magnitude below. This appears to confirm the role of nuclear shrinking (Fig. 1B) on the increase in turbidity of nuclear suspensions at Mg 2/ concentrations below 2 1 10 03 M. A direct effect of Mg 2/ ions on nuclear membrane bilayers, however, does not appear to influence the observations in Fig. 1: only a small effect, in the sense opposite to what was found for nuclei, could be detected when liposomes were dispersed, at constant osmotic pressure, in solutions of increasing Mg 2/ concentration (Fig. 2B). The observed increase in turbidity is probably due to Mg 2/ -induced liposome aggregation (10). Suspensions of isolated nuclei were subjected to thermal treatment (65 and 797C). Experiments in various media (standard buffer, 5 mM Tris, 5 mM KCl, 200 mM KCl, or 200 mM sucrose) display a common trend: temperature induces an increase ( Éthreefold) in turbidity, attributable to thermal denaturation of chromatin, as well as a decrease ( É25%) in the average nuclear size (data not shown). The effect of a nonionic solute, e.g., sucrose, on the turbidity of mouse liver nuclear suspensions was specifically examined in the presence of high (5 1 10 03 M) and low (6 1 10 05 M) Mg 2/ concentrations (Fig. 3A). Magnesium is seen to produce the characteristic increase in turbidity. Sucrose appears to have little, if any, effect on the turbidity of isolated nuclei, up to 10 01 M. A decrease in turbidity observed above that concentration is opposite to what would be expected at high osmotic pressures, if nuclei were to behave as ideal osmometers, i.e., showing nuclear shrinking
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FIG. 2. Effect of magnesium ions on the turbidity of nuclear components in suspension. (A) Isolated chromatin in 0.1 mM PMSF, 19 mM EDTA, 5 mM Tris–HCl buffer, pH 7.4, was transferred into different MgCl2 solutions and incubated for 15 min prior to the measurements. (B) Multilamellar liposomes composed of egg phosphatidylcholine:dicetylphosphate (10:1 mol ratio) were treated as indicated in Fig. 1 for nuclei under conditions of constant osmolality.
and correspondingly increasing the turbidity (11, 12). Considering that light scattering (thus turbidity) depends on the difference in refractive index between the scattering particle and the surrounding medium and that sucrose may be altering significantly the refraction index of the buffer, the influence of this factor was examined by measuring the turbidity of solid polystyrene microspheres in various sucrose solutions (Fig. 3A, triangles). However, no measurable effects were detected. Multilamellar vesicles of egg phosphatidylcholine:dicetylphosphate (10:1 mol ratio) have been used in an extensive
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PRADO ET AL.
effect, and liposome suspension turbidity decreased, as seen with the intact nuclei (Fig. 3A) under similar conditions. DISCUSSION
FIG. 3. Turbidity of mouse liver nuclei and liposomal suspensions. Turbidity was measured as absorbance at 540 nm. (A) Nuclei, (B) liposomes. Nuclei or liposomes in standard buffer (see Methods) were transferred into different sucrose solutions and incubated for 5 min at room temperature before the measurements. Experiments performed with ( l ) or without ( s ) 5 mM Mg 2/ in the sucrose solution. ( , ) Polystyrene microspheres treated as above.
series of studies on liposome permeability by de Gier et al. (1, 2), in which they were shown to behave as ideal osmometers. In order to test the relative contribution of putative osmotic effects to the observed changes in absorbance, a suspension of multilamellar vesicles of similar turbidity as a suspension of nuclei in standard buffer was treated with increasing sucrose concentrations (Fig. 3B). Irrespective of Mg 2/ concentrations, increasing the outer osmotic pressure by sucrose addition produced the expected increase in turbidity, usually attributed to vesicle shrinkage, up to a 10 01 M sucrose concentration. Above it, sucrose had the opposite
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The present work was intended to clarify the origin of the complex changes observed in the turbidity of nuclear suspensions in the presence of certain solutes (3). Turbidity measurements have been extensively used in the study of osmotically-induced volume changes in vesicles surrounded by either cell (11, 12) or model (1, 2, 13) membranes. In these cases, the transfer of vesicles to a hypotonic or hypertonic medium containing a nonpermeant species leads, respectively, to vesicle swelling or shrinking; this, in turn, is reflected as a decrease or increase in turbidity. The so-called turbidity, often measured as absorbance at a wavelength for which no chromophores exist in the sample, is in fact a consequence of light scattering by the particles (vesicles) in suspension. As seen in Figs. 1A and 3A, the turbidity of isolated nuclei in suspension does not appear to follow the expected osmotically driven changes in turbidity. However, changes in the concentration of Mg 2/ induce marked changes in turbidity (Fig. 1A). Interpretation of these results requires taking into account the origin of light scattering. In our case, light scattering is produced by particles larger than the incident light wavelength, i.e., the simple Rayleigh scattering conditions do not apply. Thus, in our case, light scattering is a function of many factors, including reflection, diffraction, refraction, and interference (2, 13, 14). Also, nuclei being inhomogeneous particles, changes in internal scattering (15, 16) are also important, internal homogeneity leading generally to a decreased scattering. In particular, in our work we have identified the following factors contributing to the observed turbidity: chromatin condensation, nuclear size, and changes in the optical properties of the membrane bilayer. Changes in the chromatin condensation as a function of cation concentration have been repeatedly observed (3–5, 17); our results (Figs. 1A and 2A) confirm that this may be at the origin of the main turbidity changes. Chromatin condensation increases particle inhomogeneity, thus increasing the turbidity. Thermal denaturation of chromatin, leading to internal inhomogeneity, is probably also at the origin of observed thermally dependent increases in turbidity (data not shown). Nuclear size decreases concomitantly with an increase in turbidity, in the presence of Mg 2/ ions (Fig. 1B). The phenomenon is probably similar to that observed by Wunderlich et al. (18) in Tetrahymena. The origin of the change in nuclear size is unclear; it may be secondary to chromatin condensation, chromatin being anchored to the inner face of nuclear envelopes, or to chromatin denaturation, but it does not appear to be osmotic in origin. However, irrespective of its origin, the reduction in nuclear size will surely contribute to the increased turbidity
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TURBIDITY OF CELL NUCLEI IN SUSPENSION
(11, 12), particularly below 2 1 10 03 M Mg 2/ (see data in Fig. 2A). Finally, both Mg 2/ and sucrose appear to have nonosmotic effects on the turbidity of membrane suspensions. The increase in liposome turbidity detected at high Mg 2/ concentrations (Fig. 2B) and the opposite effect shown by sucrose above 10 01 M (Fig. 3B) probably reflect changes in phospholipid conformation within the bilayer; in one case because of electrostatic binding to polar headgroups, and in the other because of changes in the environmental polarity. Changes in phospholipid or bilayer conformation (e.g., gel–fluid transitions) are easily detected through changes in turbidity (19). For sucrose, the observed decrease in turbidity of liposome suspensions would be enough to explain the corresponding effect as detected in nuclei. The permeability of nuclear envelopes is currently attracting the attention of an increasing number of workers. While the nuclear envelope was once thought to offer little or no resistance to the movement of molecules into the nucleus (20), findings that ions (21–23) and small proteins (24, 25) do not diffuse freely into the nucleus suggest that the former views on nuclear envelope permeability may be questioned (26). In this context, the suggestion that the nuclear pores may not represent permanent discontinuities of the envelope membrane is also of significance (27–29). The above data are most easily explained by assuming that small solutes can freely permeate the nuclear envelopes in our preparations. However, any attempt to extrapolate these data to interpret the in vivo nuclear envelope permeability processes must take into account, in addition to these factors, other possible artifacts related to the anatomic preservation of the nuclear membranes following the processes of tissue homogenization and maintenance in physiological solutions.
ACKNOWLEDGMENTS This work was supported in part with funds from DGICYT (PB91-0441 and PB92-0438), University of the Basque Country (EB014/92 and E108/ 91), and the Basque Government (PGV92/03).
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REFERENCES 1. De Gier, J., Mandersloot, J. G., and Van Deenen, L. L. M., Biochim. Biophys. Acta 150, 666 (1986). 2. De Gier, J., Mandersloot, J. G., Hupkes, J. V., McElhaney, R. N., and Van Beek, W. P., Biochim. Biophys. Acta 233, 610 (1971). 3. Aaronson, R. P., and Woo, E., J. Cell Biol. 90, 181 (1981). 4. Widom, J., J. Mol. Biol. 190, 411 (1986). 5. Olins, D. E., and Olins, A. L., J. Cell. Biol. 53, 715 (1972). 6. Kaufman, S. H., Gibson, W., and Shaper, J. H., J. Biol. Chem. 258, 2710 (1983). 7. Kay, R. R., Fraser, D., and Johnston, I. R., Eur. J. Biochem. 30, 145 (1972). 8. Laval, M., and Bouteille, M., Exp. Cell. Res. 76, 337 (1973). 9. Koch, A. L., Biochim. Biophys. Acta 51, 429 (1961). 10. Bentz, J., Du¨zgu¨nes, N., and Nir, S., Biochemistry 22, 3320 (1983). 11. Packer, L., J. Biol. Chem. 236, 214 (1961). 12. Gon˜i, F. M., Gondra, M. A., Gurtubay, J. I. G., and Macarulla, J. M., Int. J. Biochem. 11, 507 (1980). 13. Prado, A., da Costa, M. S., and Madeira, V. M. C., Int. J. Biochem. 22, 1497 (1990). 14. Bryant, F. D., Latimer, P., and Seiber, B. A., Arch. Biochem. Biophys. 135, 109 (1969). 15. Pittz, E. P., Lee, J. C., Bablouzian, B., Townend, R., and Timascheff, S. N., Methods Enzymol. 27, 209 (1973). 16. Latimer, P., Moore, D. D., and Bryant, F. D., J. Theor. Biol. 21, 348 (1968). 17. Engelhardt, M., Biochim. Biophys. Acta 1087, 173 (1990). 18. Wunderlich, F., Giese, G., and Bucherer, C., J. Cell Biol. 79, 479 (1978). 19. Aranda, F. J., and Go´mez-Ferna´ndez, J. C., Biochim. Biophys. Acta 820, 19 (1985). 20. Paine, P. L., and Horowitz, S. B., in ‘‘Cell Biology: A Comprehensive Treatise’’ (D. M. Prescott and L. Goldstein, Eds.), Vol. 4. Academic Press, New York, 1980. 21. Mazzanti, M., De Felice, L. J., Cohen, J., and Walton, H., Nature 343, 764 (1990). 22. Neylon, C. B., Hoyland, J., Mason, W. T., and Irvine, R., Am. J. Physiol. 259, c675 (1990). 23. Bustamante, J. O., Eur. J. Phys. 421, 473 (1992). 24. Breeuer, M., and Goldfarb, D. G., Cell 60, 999 (1990). 25. Ham, J., and Parker, M. G., Curr. Op. Cell. Biol. 1, 503 (1989). 26. Dingwall, C., BioEssays 13, 213 (1991). 27. Are´chaga, J., and Bahr, G. F., in ‘‘Nuclear Envelope Structure and RNA Maturation’’ (E. A. Semuckler and G. A. Clawson, Eds.), p. 23. A. R. Liss, New York, 1985. 28. Are´chaga, J., Dı´az, J., and Bahr, G. F., Anal. Quant. Cytol. Histol. 9, 335 (1987). 29. Are´chaga, J., Dı´az, J., Silio´, M., and Bahr, G. F., Biol. Cell 68, 13 (1990).
AP: Colloid
177, 9–13 (1996)
Article No. 0002
The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering ADELINA PRADO,† CARMEN PUYO,* JON ARLUCEA,* FELIX M. GON˜I,† ,1
AND
JUAN ARECHAGA *
Departments of *Cell Biology and †Biochemistry, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain Received September 27, 1994; accepted May 22, 1995
The movement of water and solutes across membranes in closed vesicles is most conveniently measured following changes in suspension turbidity (1, 2). However, the complex structure of isolated nuclei makes it difficult to relate turbidity changes to changes in volume, in turn attributable to flow of water and/or solutes. Several reports have described turbidity changes due to cation-dependent chromatin condensation (3–5), while the effects of osmotic changes on turbidity and nuclear volume have not been systematically studied, to our knowledge. In order to clarify, at least partially, the origin of changes in the turbidity of nuclear suspensions, we have studied the effect of various ionic and nonionic solutes on isolated nuclei and artificial phospholipid vesicles (liposomes), combining turbidimetric measurements with light-microscopy estimations of nuclear size. We conclude that, apart from chromatin condensation, changes in nuclear size and changes in bilayer structure or phospholipid conformation may also contribute to the observed changes in turbidity of the nuclear suspensions.
The turbidity of a suspension of cell nuclei isolated from animal tissue homogenates is a complex case of non-Rayleigh scattering. As a first approximation to this system, we have characterized a number of factors that may contribute to the observed turbidity: cation-dependent chromatin condensation, thermal denaturation of chromatin, nuclear shrinking, and changes in the optical properties of the membrane bilayer. Small differences in cation concentration, particularly in the case of divalent cations, lead to large changes in chromatin supramolecular organization, thus to large turbidity effects; thermally-induced changes in turbidity have a similar origin, although they are less pronounced. Under certain circumstances, either salts or heat may induce condensation of chromatin, the latter being connected to the inner side of the nuclear envelope, nuclear shrinking ensues, and this in turn modifies the suspension turbidity. Finally, changes in the physical properties of the lipid bilayers or of the phospholipids in the nuclear envelopes may also have significant effects, though smaller than chromatin changes, in the overall turbidity of the nuclear suspension. q 1996 Academic Press, Inc. Key Words: nucleus, cell; organelles, cell; chromatin; bilayers, lipid; light scattering.
MATERIALS AND METHODS INTRODUCTION
Mouse liver nuclei were isolated as in (6); purity was checked by microscopic, chemical, and enzymatic methods and found to be at least comparable to that of other preparations (7, 8). Turbidity was measured at room temperature, as the apparent absorbance at 540 nm, in a PW 8625 UV/ Vis Philips spectrophotometer. This is a commonly used wavelength for measuring the turbidity of subcellular organelle suspensions (9); it is low enough to produce a convenient intensity of scattering and biomolecules do not absorb strongly at this wavelength. In our case, a linear relation was observed between 540 nm scattered light and nuclear concentration, in the range 0.20–0.85 absorbance units. Protein concentration in the cuvette was 0.7 mg/ml. Nuclear sizes were evaluated by light microscopy, using a Leitz Labovert inverted microscope. The unstained nuclei were observed by the phase-contrast technique, with a magnification of 401. Nuclear diameters were measured with a standard grating ruled at normal angle and calibrated with latex micro-
Light scattering by large, heterogeneous particles presents considerable theoretical and practical problems for its analysis. Yet common use is made of light scattering or ‘‘turbidity’’ measurements in biological studies of subcellular organelles and even whole cells in suspension, particles unavoidably large and heterogeneous from the point of view of Rayleigh and related approximations. One such case is presented by the suspensions of isolated nuclei from plant or animal cells: nuclei are approximately spherical vesicles, with a diameter in the 1-mm range, limited by two membranes (each consisting of a lipid bilayer), and containing an aqueous dispersion of chromatin, a mixture of nucleic acids and proteins. The present work constitutes a first approximation to the analysis of the various factors involved in the turbidity of nuclear suspensions. 1
To whom correspondence should be addressed. 9
/ m4147$3860
11-29-95 17:08:11
coida
0021-9797/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP: Colloid
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PRADO ET AL.
spheres (4.2, 6.44, and 9.6 mm) (Polysciences, Inc. Polybead–Polystyrene Microspheres). For each data point 100 nuclei were measured. Unless otherwise stated, nuclei were resuspended in 5 mM Tris–HCl, pH 7.4, 1.73 mM sucrose, 0.6 mM KCl, 0.06 mM MgCl2 (standard buffer). For thermal treatments nuclei were incubated for 15 min at the desired temperature, then left to equilibrate at room temperature before the measurements. Osmolalities were measured in a Osmomat 030 (Gonotec). Polystyrene microspheres were from Polysciences, Inc. Multilamellar vesicles (liposomes) composed of eggyolk phosphatidylcholine and dicetylphosphate (10:1 mol ratio) were prepared and osmotically tested as in (1, 2). Chromatin was isolated from mouse liver nuclei as described in (4). Briefly, a sediment of isolated nuclei was resuspended to a concentration of 5 1 10 8 nuclei/ml in 10 mM triethanolamine/HCl, 10% (w/v) sucrose, 0.1 mM MgCl2 , pH 7.4 buffer and incubated with DNase I (25 mg/ml) at 377C for 15 min. The digestion was stopped by adding a threefold excess of ‘‘stop buffer’’ (10 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl2 , 0.5 mM PMSF, pH 7.5) at 47C. The suspension was centrifuged at 5000 1 g, 47C, 10 min, and the clear supernatant was discarded. The resulting sediment was then resuspended in four volumes of ‘‘lysis buffer’’ (10 mM Tris–HCl, 1 mM Na3EDTA, 0.1 mM PMSF, pH 7.5) and centrifuged at 20000 1 g, 47C, 10 min. The supernatant, containing the chromatin, was diluted until an A540 of É0.35 was obtained in the presence of 10 mM MgCl2 . RESULTS
Among the various cations that are known to produce changes in the turbidity of nuclei in suspension, magnesium was selected as the main divalent cation in our study because of its known affinity for the phosphate groups of nucleotides and nucleic acids. The effect of magnesium ions on the turbidity of isolated mouse liver nuclei in suspension is shown in Fig. 1A. Magnesium, in the 10 04 –10 02 M concentration range, increases the suspension turbidity. Light-microscopy observations ensure that nuclei do not aggregate in the presence of Mg 2/ in our preparations (data not shown). This experiment was first performed by adding increasing concentrations of MgCl2 , i.e., varying simultaneously Mg 2/ concentration and osmotic pressure. Both factors have then been separately studied, by repeating the experiment at increasing or constant (sucrose-compensated) osmotic pressure (Fig. 1). Changes in turbidity (Fig. 1A) are virtually the same either at constant or variable osmotic pressure. Examining the data in the light of osmolality measurements (Figs. 1A and 1C) it is seen that a large increase in turbidity occurs between 10 04 and 2 1 10 03 M Mg 2/ , while the corresponding increase in osmolality is negligible, thus confirming the idea that changes in turbidity are unrelated to changes in osmotic pressure. However, the data
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FIG. 1. Effect of magnesium ions on nuclear suspension turbidity and nuclear size. Nuclei in standard buffer (see Methods) were transferred into different MgCl2 solutions and incubated for 5 min at room temperature prior to the measurements. Experiments were carried out keeping constant the osmolality of the MgCl2 solutions, by substituting sucrose for MgCl2 (filled symbols) or under conditions of varying osmolality (open symbols).
in Fig. 1B reveal that the observed increase in turbidity is concomitant with a decrease in nuclear size; nuclear shrinking may certainly be responsible, either fully or in part, for the said increase in turbidity.
AP: Colloid
TURBIDITY OF CELL NUCLEI IN SUSPENSION
Above 7 1 10 02 M Mg 2/ , nuclear turbidity falls precipitously. Microscopic controls of the morphology of nuclei in suspension failed to reveal any significant nuclear disruption at these high Mg 2/ concentrations that could give rise to a decrease in turbidity. Other divalent cations (Ca 2/ , Mn 2/ ) had exactly the same effects as Mg 2/ , at similar concentrations, and unrelated to osmotic pressure. Monovalent cations (Li / , Na / , K / , Cs / ) and Tris also produced an increase in turbidity, but at higher concentrations ( ú10 02 M), that was followed by an abrupt decrease beyond 0.2 M (data not shown). No differences between individual monovalent cations were observed. In an effort to dissect the various sources of nuclear suspension turbidity, the effect of increasing Mg 2/ concentrations was separately tested on two different systems; these may be considered to represent the two main nuclear components from the point of view of turbidity, namely isolated chromatin (Fig. 2A) and chromatin-free phospholipid vesicles (Fig. 2B), the latter mimicking the nuclear envelopes. The effect of Mg 2/ on isolated chromatin reproduces the main features of its effect on isolated nuclei, particularly the increase and, later, abrupt decrease in turbidity (Figs. 1A and 2A). Note that chromatin concentration is the same in both experiments. However, for free chromatin turbidity increases significantly only above 2 1 10 03 M Mg 2/ , while a comparable effect is caused on intact nuclei by cation concentrations one order of magnitude below. This appears to confirm the role of nuclear shrinking (Fig. 1B) on the increase in turbidity of nuclear suspensions at Mg 2/ concentrations below 2 1 10 03 M. A direct effect of Mg 2/ ions on nuclear membrane bilayers, however, does not appear to influence the observations in Fig. 1: only a small effect, in the sense opposite to what was found for nuclei, could be detected when liposomes were dispersed, at constant osmotic pressure, in solutions of increasing Mg 2/ concentration (Fig. 2B). The observed increase in turbidity is probably due to Mg 2/ -induced liposome aggregation (10). Suspensions of isolated nuclei were subjected to thermal treatment (65 and 797C). Experiments in various media (standard buffer, 5 mM Tris, 5 mM KCl, 200 mM KCl, or 200 mM sucrose) display a common trend: temperature induces an increase ( Éthreefold) in turbidity, attributable to thermal denaturation of chromatin, as well as a decrease ( É25%) in the average nuclear size (data not shown). The effect of a nonionic solute, e.g., sucrose, on the turbidity of mouse liver nuclear suspensions was specifically examined in the presence of high (5 1 10 03 M) and low (6 1 10 05 M) Mg 2/ concentrations (Fig. 3A). Magnesium is seen to produce the characteristic increase in turbidity. Sucrose appears to have little, if any, effect on the turbidity of isolated nuclei, up to 10 01 M. A decrease in turbidity observed above that concentration is opposite to what would be expected at high osmotic pressures, if nuclei were to behave as ideal osmometers, i.e., showing nuclear shrinking
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FIG. 2. Effect of magnesium ions on the turbidity of nuclear components in suspension. (A) Isolated chromatin in 0.1 mM PMSF, 19 mM EDTA, 5 mM Tris–HCl buffer, pH 7.4, was transferred into different MgCl2 solutions and incubated for 15 min prior to the measurements. (B) Multilamellar liposomes composed of egg phosphatidylcholine:dicetylphosphate (10:1 mol ratio) were treated as indicated in Fig. 1 for nuclei under conditions of constant osmolality.
and correspondingly increasing the turbidity (11, 12). Considering that light scattering (thus turbidity) depends on the difference in refractive index between the scattering particle and the surrounding medium and that sucrose may be altering significantly the refraction index of the buffer, the influence of this factor was examined by measuring the turbidity of solid polystyrene microspheres in various sucrose solutions (Fig. 3A, triangles). However, no measurable effects were detected. Multilamellar vesicles of egg phosphatidylcholine:dicetylphosphate (10:1 mol ratio) have been used in an extensive
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effect, and liposome suspension turbidity decreased, as seen with the intact nuclei (Fig. 3A) under similar conditions. DISCUSSION
FIG. 3. Turbidity of mouse liver nuclei and liposomal suspensions. Turbidity was measured as absorbance at 540 nm. (A) Nuclei, (B) liposomes. Nuclei or liposomes in standard buffer (see Methods) were transferred into different sucrose solutions and incubated for 5 min at room temperature before the measurements. Experiments performed with ( l ) or without ( s ) 5 mM Mg 2/ in the sucrose solution. ( , ) Polystyrene microspheres treated as above.
series of studies on liposome permeability by de Gier et al. (1, 2), in which they were shown to behave as ideal osmometers. In order to test the relative contribution of putative osmotic effects to the observed changes in absorbance, a suspension of multilamellar vesicles of similar turbidity as a suspension of nuclei in standard buffer was treated with increasing sucrose concentrations (Fig. 3B). Irrespective of Mg 2/ concentrations, increasing the outer osmotic pressure by sucrose addition produced the expected increase in turbidity, usually attributed to vesicle shrinkage, up to a 10 01 M sucrose concentration. Above it, sucrose had the opposite
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The present work was intended to clarify the origin of the complex changes observed in the turbidity of nuclear suspensions in the presence of certain solutes (3). Turbidity measurements have been extensively used in the study of osmotically-induced volume changes in vesicles surrounded by either cell (11, 12) or model (1, 2, 13) membranes. In these cases, the transfer of vesicles to a hypotonic or hypertonic medium containing a nonpermeant species leads, respectively, to vesicle swelling or shrinking; this, in turn, is reflected as a decrease or increase in turbidity. The so-called turbidity, often measured as absorbance at a wavelength for which no chromophores exist in the sample, is in fact a consequence of light scattering by the particles (vesicles) in suspension. As seen in Figs. 1A and 3A, the turbidity of isolated nuclei in suspension does not appear to follow the expected osmotically driven changes in turbidity. However, changes in the concentration of Mg 2/ induce marked changes in turbidity (Fig. 1A). Interpretation of these results requires taking into account the origin of light scattering. In our case, light scattering is produced by particles larger than the incident light wavelength, i.e., the simple Rayleigh scattering conditions do not apply. Thus, in our case, light scattering is a function of many factors, including reflection, diffraction, refraction, and interference (2, 13, 14). Also, nuclei being inhomogeneous particles, changes in internal scattering (15, 16) are also important, internal homogeneity leading generally to a decreased scattering. In particular, in our work we have identified the following factors contributing to the observed turbidity: chromatin condensation, nuclear size, and changes in the optical properties of the membrane bilayer. Changes in the chromatin condensation as a function of cation concentration have been repeatedly observed (3–5, 17); our results (Figs. 1A and 2A) confirm that this may be at the origin of the main turbidity changes. Chromatin condensation increases particle inhomogeneity, thus increasing the turbidity. Thermal denaturation of chromatin, leading to internal inhomogeneity, is probably also at the origin of observed thermally dependent increases in turbidity (data not shown). Nuclear size decreases concomitantly with an increase in turbidity, in the presence of Mg 2/ ions (Fig. 1B). The phenomenon is probably similar to that observed by Wunderlich et al. (18) in Tetrahymena. The origin of the change in nuclear size is unclear; it may be secondary to chromatin condensation, chromatin being anchored to the inner face of nuclear envelopes, or to chromatin denaturation, but it does not appear to be osmotic in origin. However, irrespective of its origin, the reduction in nuclear size will surely contribute to the increased turbidity
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(11, 12), particularly below 2 1 10 03 M Mg 2/ (see data in Fig. 2A). Finally, both Mg 2/ and sucrose appear to have nonosmotic effects on the turbidity of membrane suspensions. The increase in liposome turbidity detected at high Mg 2/ concentrations (Fig. 2B) and the opposite effect shown by sucrose above 10 01 M (Fig. 3B) probably reflect changes in phospholipid conformation within the bilayer; in one case because of electrostatic binding to polar headgroups, and in the other because of changes in the environmental polarity. Changes in phospholipid or bilayer conformation (e.g., gel–fluid transitions) are easily detected through changes in turbidity (19). For sucrose, the observed decrease in turbidity of liposome suspensions would be enough to explain the corresponding effect as detected in nuclei. The permeability of nuclear envelopes is currently attracting the attention of an increasing number of workers. While the nuclear envelope was once thought to offer little or no resistance to the movement of molecules into the nucleus (20), findings that ions (21–23) and small proteins (24, 25) do not diffuse freely into the nucleus suggest that the former views on nuclear envelope permeability may be questioned (26). In this context, the suggestion that the nuclear pores may not represent permanent discontinuities of the envelope membrane is also of significance (27–29). The above data are most easily explained by assuming that small solutes can freely permeate the nuclear envelopes in our preparations. However, any attempt to extrapolate these data to interpret the in vivo nuclear envelope permeability processes must take into account, in addition to these factors, other possible artifacts related to the anatomic preservation of the nuclear membranes following the processes of tissue homogenization and maintenance in physiological solutions.
ACKNOWLEDGMENTS This work was supported in part with funds from DGICYT (PB91-0441 and PB92-0438), University of the Basque Country (EB014/92 and E108/ 91), and the Basque Government (PGV92/03).
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