A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins

Rickard Frost,Delphine Débarre,Jürgen Schünemann,Saikat Jana,Ralf P Richter,Fouzia Bano,Dirk Görlich

doi:10.1021/acsnano.0c02895

Abstract

We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane–sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine–glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.

Highlights

We introduce plane−sphere confinement microscopy with the purpose to allow studies of diffusion processes within solvated polymer films at the solid−liquid interface
A planar and a semispherical surface, both functionalized with the polymer film of interest, were brought into contact in a well-controlled fashion using a micromanipulator (Figure 2A)
Close to the point of contact between the planar and spherical surface the polymer films will overlap, excluding all bulk liquid. This is the region of primary interest for plane−sphere confinement microscopy (PSCM): thanks to the large size of the hemisphere, its lateral dimensions will exceed 10 μm for polymer coatings of >10 nm in thickness. Processes on this length scale can be readily resolved by fluorescence microscopy, enabling the characterization of diffusion processes inside the polymer film without interference from the bulk solution

Summary

A RECONSTITUTED MODEL OF THE NUCLEAR PORE PERMSELECTIVITY BARRIER

Films of end-grafted FG domains (such as FG domain brushes)[3,32,33] have been successfully used as a model system to study the properties and mechanisms of function of the nuclear pore permselectivity barrier. We use PSCM to extract information regarding both partitioning and diffusion of probe molecules within FG domain films. As probe molecules we utilize enhanced green fluorescent protein (GFPStd), a GFP mutant designed not to bind to FG domains (GFPInert), and a GFP mutant designed to gain NTR-like properties (GFPNTR).[37] We demonstrate the use of PSCM and quantify the diffusion coefficient of an NTR-like protein within an ultrathin film of FG domains

RESULTS AND DISCUSSION

CONCLUSIONS

MATERIALS AND METHODS