Stable, non-destructive immobilization of native nuclear membranes to micro-structured PDMS for single-molecule force spectroscopy.
In eukaryotic cells the nucleus is separated from the cytoplasm by a double-membraned nuclear envelope (NE). Exchange of molecules between the two compartments is mediated by nuclear pore complexes (NPCs) that are embedded in the NE membranes. The translocation of molecules such as proteins and RNAs through the nuclear membrane is executed by transport shuttling factors (karyopherines). They thereby dock to particular binding sites located all over the NPC, the so-called phenylalanine-glycin nucleoporines (FG Nups). Molecular recognition force spectroscopy (MRFS) allows investigations of the binding at the single-molecule level. Therefore the AFM tip carries a ligand for example, a particular karyopherin whereas the nuclear membrane with its receptors is mounted on a surface. Hence, one of the first requirements to study the nucleocytoplasmatic transport mechanism using MRFS is the development of an optimized membrane preparation that preserves structure and function of the NPCs. In this study we present a stable non-destructive preparation method of Xenopus laevis nuclear envelopes. We use micro-structured polydimethylsiloxane (PDMS) that provides an ideal platform for immobilization and biological integrity due to its elastic, chemical and mechanical properties. It is a solid basis for studying molecular recognition, transport interactions, and translocation processes through the NPC. As a first recognition system we investigate the interaction between an important transport shuttling factor, importin beta, and its binding sites on the NPC, the FG-domains.
Rangl
Martina
M
Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, 4040 Linz, Austria.
Nevo
Reinat
R
Liashkovich
Ivan
I
Shahin
Victor
V
Reich
Ziv
Z
Ebner
Andreas
A
Hinterdorfer
Peter
P
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009685:Nuclear Envelope / Q000737:chemistry* / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry / Q000378:metabolism; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D004129:Dimethylpolysiloxanes; D028961:beta Karyopherins; C013830:baysilon