ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope

Rakowska, Agnieszka; Danker, Timm; Sw, Schneider; Oberleithner, Hans

doi:10.1007/s002329900377

Cited by 82 publications

(59 citation statements)

References 33 publications

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“…Our prolonged patch-clamp recordings (1-36 h) from fully functional nuclei do not contradict the AFM observation of Ca 2+ -dependent NPC plugs (see [4,5,6]) or ATPdependent NPC geometry [21] because the NPC model including an intrinsic plug must account for the ease of diffusion of monoatomic ions along the NPCC. That is, the plug must have a hole sufficiently large to account for the ion flow always seen in isolated and in situ nuclei.…”

Section: Ca 2+ and The Npc Plugmentioning

confidence: 43%

“…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). To date, no-one has shown that NPC channels (NPCCs) are permanently closed to monoatomic ion flow.…”

Section: Introductionmentioning

confidence: 99%

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Calcium, ATP and nuclear pore channel gating

Bustamante

Michelette

Geibel

et al. 2000

Pfl�gers Archiv European Journal of Physiology

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Nuclear envelope (NE) cisternal Ca 2+ and cytosolic ATP are required for nuclear-pore-complex-(NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (γ) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca 2+ nor ATP alone directly affects NPCC gating. To test the role of Ca 2+ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from HHS Public Access

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Section: Ca 2+ and The Npc Plugmentioning

confidence: 43%

Section: Introductionmentioning

confidence: 99%

Calcium, ATP and nuclear pore channel gating

Bustamante

Michelette

Geibel

et al. 2000

Pfl�gers Archiv European Journal of Physiology

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“…The finding that the majority of the NPCs obviously are in an electrically open state with a high electrical conductance of 1.7 nS per NPC is in apparent contradiction to patch clamp experiments that show gigaseals lacking such large conductances (10). This striking differences cannot solely be explained by differences in the origin of the nuclei, because gigaseals lacking such large conductances also have been reported from the same type of nuclei preparation (17). The current view is that most of the NPCs are closed during patch clamp experiments (31).…”

Section: Discussionmentioning

confidence: 68%

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

Danker

Schillers

Storck

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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“…During the past several years, many laboratories have succeeded in visualizing NPCs by SFM (Pante and Aebi 1993;Braunstein and Spudich 1994;Goldie et al 1994;Oberleithner et al 1994Oberleithner et al , 1996Folprecht et al 1996;PerezTerzic et al 1996;Rakowska et al 1998;Jaggi et al 2003a,b). However, none of these examinations were conducted on fully native NPCs.…”

Section: The Current Consensus Model Of the Nuclear Pore Complexmentioning

confidence: 99%

From the trap to the basket: getting to the bottom of the nuclear pore complex

2006

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Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the doublemembraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of highresolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.

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ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope

Cited by 82 publications

References 33 publications

Calcium, ATP and nuclear pore channel gating

Calcium, ATP and nuclear pore channel gating

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

From the trap to the basket: getting to the bottom of the nuclear pore complex

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