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 M ay 5, 2017 D ow nladed fom Published July 1, 1990