This laboratory recently found that the relative rate of signal- mediated transport through the nuclear pores, as well as the functional size of the transport channel, decrease significantly when proliferating 3T3 cells enter a period of growth arrest. To better understand the role of nuclear envelope permeability in regulating cellular activity, experiments are proposed to determine 1) if the growth-related effects on transport are limited to nonspecific changes (e.g., functional pore size), or whether the specificity for different targeting signals is also affected, 2) whether the size restrictions that limit nuclear uptake in growth arrested cells also apply to nuclear efflux, 3) if the observed permeability changes are due to alterations in cell shape, modifications in the composition of the pore glycoproteins, or variations in the activity of cytoplasmic transport factors, and 4) if growth factors are involved in the modulation of signal- mediated nuclear transport. In order to analyze nuclear transport, cultured cells will be injected with either various size colloidal gold particles coated with proteins containing nuclear localization signals, or fluorescent-labeled BSA conjugated with different targeting sequences. Uptake into the nucleus will be analyzed by electron or fluorescent microscopy. Communication and protein movement between the nucleus and the cytoplasm of an interphase eukaryotic cell is crucial to the cell's ability to regulate such fundamental cellular processes as protein synthesis, gene expression, and cell cycle. Movement of proteins and nucleic acids back and forth across the nuclear envelope is known to take place at discrete specialized structures called nuclear pores. At least some materials move specifically through pores via a system of "targeting sequences" and receptors which recognize and facilitate such movement, although the detailed mechanism(s) by which these transport phenomena occur remain unknown. This project will contribute significantly to understanding of the complex nature of nuclear targeting.
