Diffusion Across the Nuclear Envelope Inhibited by Depletion of the Nuclear Ca ²⁺ Store

Abstract

Intact, isolated nuclei and a nuclear membrane (ghost) preparation were used to study regulation of the movement of small molecules across the Xenopus laevis oocyte nuclear membrane. In contrast to models of the nuclear pore complex, which assume passive bidirectional diffusion of molecules less than 70 kilodaltons, diffusion of intermediate-sized molecules was regulated by the nuclear envelope calcium stores. After depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10-kilodalton dextran were unable to enter the nucleus. Dye exclusion after calcium store depletion was not dependent on the nuclear matrix because it occurred in nuclear ghosts lacking nucleoplasm. Smaller molecules and ions (500-dalton Lucifer yellow and manganese) diffused freely into the core of the nuclear ghosts and intact nuclei even after calcium store depletion. Thus, depletion of the nuclear calcium store blocks diffusion of intermediate-sized molecules.

Calcium, ATP and nuclear pore channel gating

“…Recent independent patch-clamp and fluorescence microscopy studies confirm this view [16,17]. That is, nuclei isolated at our laboratory (e.g., [18,19]) and elsewhere (e.g., [15]) show that the NE cisterna is isolated from the extra-and intra-nuclear compartments. AFM has also been used to show that extranuclear Ca 2+ and ATP can induce changes in NPC shape ( [20] and [21], respectively).…”

“…Note that by substrate we mean a substance required for MMT and, therefore, the term does not include the translocating particle, which we will call heretofore MMT "cargo" after [37]. Ca 2+ loading of the NE cisterna as well as part of the MMT substrates were secured with the ATP-regenerating system plus 1 µM [Ca 2+ ] [15]. Here we refer to this mixture as the Ca+ATP system.…”

“…Ca 2+ loading of the NE cisterna was confirmed by imaging the NE with the membrane-permeant form of fluo-3 (fluo-3/AM, Molecular Probes). Nuclei were incubated for 15 min in 100 µM probe and then washed with control saline [15]. Note that for all fluorescent probes, except FITC-rNTPs and fluo-3/AM, the probes were left in the bath as they did not interfere with the imaging of the effect.…”

“…To date, no-one has shown that NPC channels (NPCCs) are permanently closed to monoatomic ion flow. However, in contrast, it has been shown that, at the macroscopic level, both monoatomic ions and small molecules enter the nucleus under the most unphysiological conditions (e.g., [14,15]). We showed, in a series of reports combining patch-clamp with laser scanning confocal microscopy (and supported with EM and AFM), that the NPC of isolated cardiomyocyte nuclei has an intrinsic ion channel behavior that can be transiently and reversibly silenced (i.e., the measured ion current is zeroed) by transcription factors and other nuclear macromolecules in a fashion that suggests transient plugging of the NPCC [18,22,23].…”

“…NPCs may be plugged by reducing [Ca 2+ ] NE , which explains the fluorescence and luminescence microscopy observations that, under certain conditions, the NE is capable of monoatomic ion-barrier behavior (e.g., [8,9] -reviewed in [10,11,12]). Furthermore, small dextran molecules (e.g., <10 kDa) placed outside the nucleus translocate into the nuclear interior and not into the NE cisterna [13,14,15], demonstrating that only the NPCCs, and not channels at either of the two NE membranes, provide an electrical and chemical shortcircuit between the pipette and bath electrodes in nucleus-attached patch-clamp preparations. Recent independent patch-clamp and fluorescence microscopy studies confirm this view [16,17].…”