Permeability of the Nuclear Envelope at IsolatedXenopusOocyte Nuclei Studied by Scanning Electrochemical Microscopy

Abstract

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.