Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

Abstract

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2͞3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ؎ 0.34 S͞cm 2 ). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 ؋ 10 6 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically ''open'' state with a mean single NPC electrical conductance of 1.7 ؎ 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.nucleus ͉ electrophysiology N uclear envelopes (NEs) of Xenopus laevis oocytes have been the favored preparation for evaluation of nuclear transport and for studies of nuclear pore complexes (NPCs) structure and function, but only limited data are available on the electrophysiological properties of this important barrier, mainly because of technical limitations.Electrophysiological evaluation of NE started in the early sixties. Using two microelectrodes impaling the nucleus of a Drosophila salivary gland cell, Loewenstein and his coworkers (1) measured the electrical conductance of the NE. The conductance was much smaller than they had expected. At that time, NPCs were thought to be open gaps in the nuclear membrane with a diameter of at least 40 nm. From their electrical conductance measurements, Loewenstein and coworkers concluded that a protein structure must be present in these gaps that restricts the opening to less than 10 nm, resulting in an electrical conductance of about 1 nS per nuclear pore (2). This observation came very close to the view of the nuclear pore that has emerged as a result of much later studies, in which the diffusion of differently sized molecules was measured. According to this view, the NPC forms an aqueous channel of 8-12 nm in diameter and 40-50 nm in length (3). From this data, a NPC electrical conductance of 1-2 nS can be calculated (4).In ...

Aldosterone signaling pathway across the nuclear envelope

“…The technical aspects of the method and its application in isolated cell nuclei have been described in detail (15). In short, the method uses a tapered glass tube, which narrows in its middle part to two-thirds of the diameter of the nucleus.…”

“…Electrical current through NPCs can be measured by patchclamp techniques applied to cell nuclei (11)(12)(13) and related to macromolecule translocation (14). Whole NE electrical conductance can be measured by the recently developed nuclear hourglass technique (NHT) (15) and related to NPC function.…”

Glucocorticoids remodel nuclear envelope structure and permeability

“…Using a nano-imaging technique, atomic force microscopy (AFM) (Binnig and Quate, 1986), we could visualize some of the endogeneous macromolecules on their route through the NPCs following aldosterone injection into X. laevis oocyte (Schäfer et al, 2002). Changes in NPC conformation were paralleled by sharp changes in NE electrical conductivity (Danker et al, 1999). In the present study we expressed the exogeneous rat glucocorticoid receptor in X. laevis oocytes and studied the impact of a potent glucocorticoid hormone analogue on the structure and the permeability of the NE and the NPC conformation.…”

Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore

“…Moreover, there is increasing evidence that the receptor-mediated transport and passive diffusion of ions and small macromolecules occurs via peripheral routes in the NPC, presumably, the so-called peripheral channels, whose presence has been initially postulated by electron microscopy in 1992 [11]. Support for the existence of peripheral channels in the NPC comes from later studies investigating the NPC both from structural and functional aspects using permeability investigating approaches and the nano-imaging technique atomic force microscopy (AFM), respectively [12][13][14][15]. AFM not only yields extraordinary high-resolution images [16] but can also be used as a nano-mechanical tool for studying numerous properties of the imaged sample such as hydrophobicity [17,18].…”

“…The technical aspects of the method and its application to isolated cell nuclei have previously been described in detail [12,19]. In brief, the method uses a tapered glass tube that narrows in the middle to two thirds the diameter of the nucleus.…”