Steroids dilate nuclear pores imaged with atomic force microscopy.
Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC-free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.
Shahin
Victor
V
Nanolab, Institute of Physiology II, University of Münster, Münster, Germany. shahin@uni-muenster.de
Albermann
Lars
L
Schillers
Hermann
H
Kastrup
Lilian
L
Schäfer
Claudia
C
Ludwig
Yvonne
Y
Stock
Christian
C
Oberleithner
Hans
H
D000818:Animals; D001692:Biological Transport; D003907:Dexamethasone / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005260:Female; D005938:Glucocorticoids / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006727:Hormone Antagonists / Q000494:pharmacology; D007267:Injections; D018625:Microscopy, Atomic Force; D015735:Mifepristone / Q000494:pharmacology; D022022:Nuclear Pore / Q000187:drug effects* / Q000502:physiology* / Q000648:ultrastructure; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors; D014982:Xenopus laevis
D005938:Glucocorticoids; D006727:Hormone Antagonists; D011506:Proteins; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D003907:Dexamethasone