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Stable, Non‐Destructive Immobilization of Native Nuclear Membranes to Micro‐Structured PDMS for Single‐Molecule Force Spectroscopy
Author(s) -
Rangl Martina,
Nevo Reinat,
Liashkovich Ivan,
Shahin Victor,
Reich Ziv,
Ebner Andreas,
Hinterdorfer Peter
Publication year - 2009
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200900219
Subject(s) - membrane , nuclear pore , force spectroscopy , chemistry , biophysics , nuclear transport , polydimethylsiloxane , molecule , nucleoporin , cytoplasm , molecular recognition , nanotechnology , cell nucleus , materials science , biochemistry , biology , organic chemistry
Stable anchorage: Micro‐structured polydimethylsiloxane serves as an optimal specimen‐support for immobilizing native nuclear membranes. It offers a solid basis for studying single‐molecule interaction of proteins involved in nucleocytoplasmatic transport (see figure).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 β, and its binding sites on the NPC, the FG‐domains.