The Permeability of the Nuclear Envelope in Dividing and Nondividing Cell Cultures

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-LI cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 ~, in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to "~230/~. T HERE are potentially two general mechanisms for controlling macromolecular exchanges between the nucleus and cytoplasm. The first is dependent on the physical and chemical properties of the permeant molecules. Modulation of these properties can affect both passive diffusion and transport across the nuclear envelope. Diffusion occurs through aqueous channels, '~100 A in diameter, that are located in the centers of the nuclear pore complexes. The rates of exchange are inversely related to molecular size, and substances larger than '~70 kD are effectively excluded from the diffusion channel (22, 23). Molecules or molecular aggregates as large as 260 A in diameter can be actively transported across the pores if they contain appropriate nuclear targeting sequences (4, 8). However, the rates of transport, as well as the size of the transport channel, are variable, and known to be dependent on both the number and the specific amino acid composition of the targeting signals (2, 4). A second possible regulatory mechanism could involve modifications of the permeability characteristics of the pore complexes, caused by changes in cellular activity. Although this concept has important functional implications, very little pertinent data is available. Jiang and Schindler (14) reported changes in the nuclear uptake of fluorescein-labeled dextrans in 3T3 cells after treatment with epidermal growth factor and insulin. Feldherr (6, 7) investigated the intracellular distribution of polyvinylpyrrolidone (PVp)l-coated gold particles in dividing amebas, and observed that the nuclei were most permeable during the first 2 h after division. Since neither dextran nor PVP contain nuclear targeting signals, these results reflect changes in passive diffusion across the envelope. Currently, there is no information regarding modifications in signal-mediated nuclear transport. The purpose of this study was to determine whether the properties of the nuclear pore complexes vary during different periods of activity in specific cell types. Both diffusion and transport of macromolecules across the envelope were analyzed throughout the HeLa cell cycle, and also in dividing, confluent (growth-arrested) and differentiated 3T3-LI cells. Colloidal gold particles coated with BSA were used to study passive diffusion. Gold coated with either nucleoplasmin or BSA conjugated with peptides containing the SV40 nuclear targeting signal was used in transport studies. The colloidal tracers were microinjected into the cytoplasm, and their subsequent intracellular distribution was determined by electron microscopy. Using this experimental approach, relative nuclear uptake, as well as the dimensions of the exchange channels, could be analyzed. 1. Abbreviations used in this paper: N/C, nuclear to cytoplasmic; PVP, polyvinylpyr rolidone. © The Rockefeller University Press, 0021-9525190107/118 $2.00 The Journal of Cell Biology, Volume 111, July 1990 1-8 1 on O cber 0, 2017 jcb.rress.org D ow nladed fom Significant variations in passive diffusion across the envelope were detected at different times in the HeLa cell cycle. A close correlation exists between the peaks of diffusion and the maximum rates of nuclear pore formation. In addition, significant differences in both the relative nuclear uptake of nucleoplasmin-gold and the dimensions of the transport channel were detected during different functional states in 3T3-LI cells. Materials and Methods Culture and Synchronization Procedures HeLa cells were maintained in "I25 flasks in 5% CO2 at 37°C and subcultured every 3 d. The culture fluid consisted of Minimum Essential Media (Alpha-Mere) containing 10% FBS, penicillin G (10,000 U/ml), streptomycin sulfate (10 mg/ml), and fungizone (250 ttg/ml). All tissue culture reagents were purchased from Gibco Laboratories (Grand Island, NY). Before microinjection of HeLa cells, mitotic shake-offs were performed as follows: cell aggregates and debris were removed from 1to 2-d-old cultures by slowly pouring off the culture medium; fresh medium was added, and the flasks were gently shaken. The dissociated cells were then plated on gridded, collagen-coated ACLAR cover slips (see below) that were attached to the bottom of 35-ram Petri dishes. 60% of the cells that were obtained by this procedure were in metaphase, anaphase, or telophase; 30% were in early interphase, as judged from their size; the remaining 10% could not be characterized. These cultures were used to study nuclear permeability at the later stages of the cell cycle (7-20 h). At earlier periods in the cycle (15 min to 5 h), greater precision was required and it was necessary to time the cells individually. To accomplish this, the shake-off cultures were grown in an incubator for "~20 h, at which time they were transferred to an inverted Nikon microscope fitted with an environmental chamber that was maintained at 37°C. Carbon dioxide was supplied to keep the pH of the cultures at 7.4, and mineral oil was added to the surface of the medium to prevent evaporation. Cells just entering anaphase (0 time) were identified, their position on the ACLAR cover slip was recorded and they were injected 15 min to 5 h later. The time required for progression of the cells through mitosis was the same as described by Robbins and Gonatus (25). 3T3-L1 celts were grown in DME supplemented with FBS, penicillin G, streptomycin, and fungizone (at concentrations given above), and kept at 37°C in 5% CO2. Subculturing was carried out as described by Frost and Lane (10); the cells were discarded after the tenth passage. Dividing, confluent, and differentiated cells to be used for nuclear permeability studies were prepared as follows: dividing populations were obtained from mitotic shake-offs of nonconfluent stock cultures. The cells were transferred to ACLAR cover slips in 35-ram Petri dishes (see below) and incubated for 8 h before microinjection. Confluent cultures were prepared by inoculating ACLAR Petri dishes with trypsinized cells at a density of 2.0 × 105 cells/ml; confluency was reached in 4-6 d. Differentiation of the confluent cells into adipocytes was accomplished by the addition of methylisobutylxanthine, dexamethasone, and insulin to the cultures (10). ACLAR (a fluoroplastic obtained from Allied Chemical) proved to be an excellent substrate for culturing and processing cells for electron microscopy (15). Before culturing, a grid pattern with ~ l m m spacing was scored on the surface of the ACLAR (18). This provided a convenient means of mapping injected and dividing cells; furthermore, since the pattern was etched on the surface of the plastic blocks after embedding, individual cells could be readily identified for electron microscopic analysis. To facilitate attachment of HeLa ceils, the ACLAR was coated with collagen as described by the supplier (Sigma Chemical Co., St. Louis, MO). Pretreatment was not required for 3T3-LI cells. Gold Preparation and Microinjection Colloidal gold fractions containing particles 20-120 A in diameter were prepared by reducing gold chloride with a saturated solution of phosphorus in ether (5). Larger particles, ranging in diameter from 80 to 240/~ and above, were obtained by using sodium citrate as a reducing agent (9). The size of the fractions varied slightly in different preparations. The gold particles were coated with BSA (Sigma Chemical Co.), nucleoplasmin, or BSA conjugated with synthetic peptides containing either active (WT) or inactive (cT) SV40 nuclear targeting signals. Nucleoplasmin was isolated from Xenopus oocytes by antibody affinity chromatography (4), and the BSA conjugates were prepared and analyzed as described in detail by Lanford et al. (16). Both the BSA-WT and BSA-cT conjugates contained an average of eight signal sequences per BSA molecule. Before injection all stabilized gold fractions were dialyzed against intracellular medium that consists of 117 mM KCI, 10.1 mM NaCI, 6 mM K2HPO4, and 4 mM KH2PO4 (pH 7.0). Unless otherwise indicated, the dimensions of the gold particles given in this report do not include the protein coat, which increases the overall particle diameter by •30 A. Microinjection was performed using an inverted microscope (Nikon Diaphot) and a Narishige hydraulic micromanipulator. The microscope was enclosed in an environmental chamber that was kept at 37°C; CO_, was provided to maintain the pH of the cultures at 7.4. The micropipets had outside tip diameters of ",,0.5-0.7 #m. A continuous flow injection system was used (11). Electron Microscope Procedures The experimental cells, still attached to ACLAR, were fxed by adding an equal volume of 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) directly to the cultures. After 30 min to 1 h at room temperature, or overnight at 4°C, the cells were rinsed in buffer, and postfixed in a mixture of 1% OsO4 and 1% K4Fe(CN)6 in 0.1 M cacodylate buffer (pH 7.3) for 1 h at room temperature. The cells were then rinsed in deionized H20, dehydrated through a graded ethanol series followed by acetone, and embedded in Spurr's low viscosity resin (26). After curing, the ACLAR was removed from the resin leaving behind the cells and an impression of the grid pattern. The cells of interest were cut out of the block, remounted, and sectioned using an MT-2 microtome fitted (Sorvall, Inc., Norwalk, CT) with a diamond knife. The sections were picked up on formvar-coated slot grids and examined using a JEOL 100CX electron microscope. To facilitate detection of the gold particles, the sections were not poststained.