A new method for in vitro studies of nucleo-cytoplasmic transport is described. The method is based on the observation mode originally by Simons and Virta ( EMBO J 6:2241-2247] that the apical plasma membrane of epithelial cells growing in monolayer culture con be mechanically removed by letting a nitrocellulose filter adsorb to the cell monolayer and then tearing it off. Using confocal imaging we find that the method con be much improved if a soft common laboratory tissue, instead of the stiff nitrocellulose filter, is employed. Cells permeabilised in that matter were found to maintain nuclear integrity with regard to passive permeability and energy- dependent protein transport. Interestingly, the nuclear envelope displayed a slightly increased passive permeability immediately after permeabilisation, which, however, spontaneously 'healed' within about 15 minutes. Permeabilised cells could be repeatedly washed without comprising nuclear envelope integrity. All together we find that mechanical permeabilisation in the version described in this paper is convenient, fast, reliable, and inexpensive.
|Number of pages||9|
|Journal||Methods in Molecular and Cellular Biology|
|Publication status||Published - 1 Dec 1994|