Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy.
A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs' nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.
Huang
Ning-Ping
NP
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China. nphuang@seu.edu.cn
Stubenrauch
Mike
M
Köser
Joachim
J
Taschner
Nicole
N
Aebi
Ueli
U
Stolz
Martin
M
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D018625:Microscopy, Atomic Force / Q000379:methods*; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
D028861:Nuclear Pore Complex Proteins