Stable, Non‐Destructive Immobilization of Native Nuclear Membranes to Micro‐Structured PDMS for Single‐Molecule Force Spectroscopy

Abstract

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 microstructured 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.

Probing High Permeability of Nuclear Pore Complexes by Scanning Electrochemical Microscopy: Ca2+ Effects on Transport Barriers

“…We employed SECM imaging to investigate the NPC-mediated transport of small redox probes across the NE spread over a microporous Si 3 N 4 membrane (Figure ). The nucleoplasm-free NE was prepared (Figure ) as established for fluorescence transport studies, which ensured the physiological function of NPCs on the NE patches in MIB, i.e., to mediate signal-dependent transport of passively impermeable large proteins by nuclear transport receptors when the proteins are labeled with nuclear localization or export signal peptides. , In this study, we employed 3-μm-diameter pores to support small patches of the NE (Figure B), which were robust enough for AFM imaging of individual NPCs (Figure ). Moreover, 3-μm-diameter pores were large enough in comparison with a 1-μm-diameter Pt tip to allow the transport of redox probes under the tip without hindrance from the pore wall (see below).…”

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture

“…The liquid PDMS was then cast against the coverslip by baking at 60 °C for at least 2 h. The resulting sheet of PDMS (~2 mm thick) was cut into a dogbone shape with a razorblade so as to fit the stretching apparatus. A 21 μg ml −1 Cell Tack solution31 was prepared in sodium bicarbonate buffer (100 mM, pH 8.3). Then, 30 μl of the solution was pipetted onto the PDMS and incubated for 15 min before being washed 15 times with HPLC grade water.…”

AFM imaging of functionalized carbon nanotubes on biological membranes

“…For such an experiment, an NPC transport protein, e.g. importin β, is needed as a bio-functional coating for CNT to study the binding to an individual NPC structure in comparison with a control sample of CNT coated with a non-binding protein [28].…”