Nuclear Plug Harvesting Using Atomic Force Microscopy

We describe an approach that aims to identify transcripts of early genes induced by the mineralocorticoid hormone aldosterone. In a first step the hormone is injected into stage VI Xenopus laevis oocytes. Then, the nuclei are mechanically isolated from the respective cells over a time period of 2 to 30 minutes after hormone injection. The isolation step interrupts the physiological hormone response of the cell at a given time point since the nucleus is transferred in electrolyte solution mimicking the cytosol but lacking metabolic substrates. Now the nuclear envelopes are manually peeled off the nuclei and spread on glass with the nucleoplasmic surface facing downwards. With atomic force microscopy (AFM) the cytoplasmic surface of the nuclear pore complexes (NPCs) are imaged and NPCs with central plugs are identified. Plugs most likely represent transcripts of aldosterone-induced early genes. For plug removal, the loading force of the AFM tip is increased until plugs get off the NPCs and become attached to the AFM tip. This procedure is called “plug harvesting”. Plugs serve as the starting matter for further mRNA analysis.