Figure 1.

Optical recording of signal-mediated protein transport through single NPCs. (A) The principle. The nuclear envelope of Xenopus oocytes is firmly attached to isoporous filters. Filter pores are sealed by mineral oil. Transport into or out of individual filter pores is measured by confocal laser scanning microscopy. (B) The transport substrates. The recombinant green fluorescent proteins IP, EP, and CP contained either a nuclear localization signal (NLS), nuclear export signal (NES), or no localization signal. (C) Import of IP. Filter pores were filled with a solution containing IP (green fluorescence), TRD70 (red fluorescence), and egg extract while a nuclear extract was applied to the unclear side. Upon addition of ATP (0 min) IP was transported out of the filter pores via the NPCs. Simultaneously, the constant TRD70 fluorescence indicated the tightness of sealing and the integrity of the NPCs. (D) Export no EP. EP was added to nuclear side and IP was omitted on the cytoplasmic side, otherwise analogous to C.