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Plug Harvesting by Atomic Force Microscopy

Abstract

Aldosterone is an important mediator of osmoregulation in vertebrate cells and is thought to be involved in cell differentation. Its signaling pathway starts with the formation of a complex with the mineralocorticoid receptor (MCR) which is then translocated into the cell nucleus where transcription of target genes is initiated. Finally, mRNAs of specifically activated genes appear in the cytoplasm. Using atomic force microscopy (AFM), we were able to visualize the signaling cascade described above. We injected stage VI oocytes of Xenopus laevis with aldosterone and isolated their nuclei after different incubation times. Nuclei obtained 2 min after application of aldosterone clearly exhibited macromolecules (“flags”) attached to the nuclear pore complexes (NPCs) when examined by AFM. The estimated size of the observed particles (80‐160 kDa) corresponds with the actual size of the MCR (∼120kDa). NPCs of oocytes injected about 20 min prior to preparation showed large macromolecules (“plugs”) within their central channels. We concluded that these plugs contain transcripts induced by aldosterone. Electrical measurements support these findings; appearance of both flags and plugs is linked to an increase in nuclear envelope electrical resistance (NEER), probably due to partial plugging of NPCs. The increase of NEER parallel to the occurrence of plugs is prevented by adding RNase A or actinomycin D (an inhibitor of transcription). Alterations of NEER and formation of flags and plugs in response to aldosterone are suppressed by coinjection of the MCR‐inhibitor spironolactone. Therefore we consider plugs to represent the early genomic response to aldosterone stimulation of Xenopus oocytes.

In conclusion, AFM not only allows imaging of structures in the submicrometer range, it may also be used to manipulate them. By applying forces to the AFM tip approximately 10 fold higher than those used for imaging we were able to dislocate plugs from NPCs (see figures). The macromolecules sticking to the AFM tip can be used as substrates for further experiments, for example RT‐PCR protocols.

Authors:   Albermann, L.; Schäfer, C.; Shahin, V.; Reinhardt, J.; Oberleithner, H.
Journal:   Single Molecules
Volume:   3
edition:   5‐6
Year:   2002
Pages:   313
DOI:   10.1002/1438-5171(200211)3:5/6<313::AID-SIMO313>3.0.CO;2-S
Publication date:   01-Nov-2002
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