Nuclear Plug Harvesting Using Atomic Force Microscopy

Oberleithner, Hans; Schäfer, Claudia; Shahin, Victor; Schlune, Andrea; Schillers, Hermann; Reinhardt, Jürgen

doi:10.1002/1438-5171(200107)2:2<117::aid-simo117>3.0.co;2-p

Cited by 7 publications

(3 citation statements)

References 14 publications

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“…We recently described a method for plug harvesting from NPCs by using the AFM as a nanosurgical tool (25). Using this technique it should be feasible to harvest plugs and decipher their mRNA by using molecular biological techniques.…”

Section: Discussionmentioning

confidence: 99%

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Aldosterone signaling pathway across the nuclear envelope

Schäfer

Shahin

Albermann

et al. 2002

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

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We describe the route by which aldosterone-triggered macromolecules enter and exit the cell nucleus of Xenopus laevis oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2-30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5-15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small (Х100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large (Х800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (i) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (ii) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (iii) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.nuclear pore complex ͉ atomic force microscopy ͉ RNA export ͉ mineralocorticoid receptor S ignals such as an increase in intracellular calcium concentration or intracellular alkalinization are the early responses of target cells to the mineralocorticoid hormone aldosterone. The intracellular calcium signal can be detected a few seconds after hormone exposure (1-3), whereas the pH signal occurs later but usually lasts longer (4). Although there are no doubts about the existence of these early hormone responses, the physiological relevance is still unknown. For almost 40 years it has been postulated that inorganic ions could play a crucial role in steroid hormone-induced gene activation (5). This suggestion has been supported by more recent findings in our own laboratory (6, 7). The signaling pathway of steroid hormones involving intracellular macromolecules is well established. Aldosterone is known to bind to mineralocorticoid receptors located in the cytosol. Hormone binding changes protein conformation and translocates the receptor into the nucleus where it attaches to specific DNA. Transcription occurs and finally mRNA is exported to be translated into aldosterone-induced proteins at the ribosomes. Although this macromolecular signal pathway has been described in detail, there are no data available that directly show both functionally and structural...

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Section: Discussionmentioning

confidence: 99%

“…1D). The latter technique (so-called plug harvesting) has been described in detail recently (25). In short, after imaging pores with plugs by using low loading forces we gradually increased force until plugs became loose.…”

Section: Methodsmentioning

confidence: 99%

Aldosterone signaling pathway across the nuclear envelope

Schäfer

Shahin

Albermann

et al. 2002

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

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“…6b) with a molecular mass of 125 MDa, and it penetrates both the ONM and the INM [149,150]. The NPC is composed of about 30 proteins named nucleoporins and forms an eightfold symmetrical structure [151][152][153][154][155]. The NPCs are likely to be newly synthesized and inserted into the nuclear envelope in the interphase cells.…”

Section: Nuclear Envelope In Interphasementioning

confidence: 99%

Nuclear architecture and chromatin dynamics revealed by atomic force microscopy in combination with biochemistry and cell biology

Hirano

Takahashi

Kumeta

et al. 2008

Pflugers Arch - Eur J Physiol

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The recent technical development of atomic force microscopy (AFM) has made nano-biology of the nucleus an attractive and promising field. In this paper, we will review our current understanding of nuclear architecture and dynamics from the structural point of view. Especially, special emphases will be given to: (1) How to approach the nuclear architectures by means of new techniques using AFM, (2) the importance of the physical property of DNA in the construction of the higher-order structures, (3) the significance and implication of the linker and core histones and the nuclear matrix/scaffold proteins for the chromatin dynamics, (4) the nuclear proteins that contribute to the formation of the inner nuclear architecture. Spatio-temporal analyses using AFM, in combination with biochemical and cell biological approaches, will play important roles in the nano-biology of the nucleus, as most of nuclear structures and events occur in nanometer, piconewton and millisecond order. The new applications of AFM, such as recognition imaging, fast-scanning imaging, and a variety of modified cantilevers, are expected to be powerful techniques to reveal the nanostructure of the nucleus.

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Nucleic Acid and Protein Single Molecule Detection and Characterization

Greulich

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Nuclear Plug Harvesting Using Atomic Force Microscopy

Cited by 7 publications

References 14 publications

Aldosterone signaling pathway across the nuclear envelope

Aldosterone signaling pathway across the nuclear envelope

Nuclear architecture and chromatin dynamics revealed by atomic force microscopy in combination with biochemistry and cell biology

Nucleic Acid and Protein Single Molecule Detection and Characterization

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