Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering

Martha Elisabeth Brennich,Thomas Pfohl,Jens-Friedrich Nolting,Christian Dammann,Harald Herrmann,Sarah Köster,Bernd Nöding,Susanne Bauch

doi:10.1039/c0lc00319k

Abstract

The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques.

Highlights

Intermediate filaments (IFs) are built from extended coiled-coil proteins and are principal components of the cytoskeleton of metazoan cells
As the flow continues downward the main channel, the salt diffuses into the vimentin solution and starts the assembly process
To prevent the adsorption of assembled vimentin to the channel walls, we seek to cover the vimentin with salt buffer from all sides as soon as it enters the intersection

Summary

Introduction

Intermediate filaments (IFs) are built from extended coiled-coil proteins and are principal components of the cytoskeleton of metazoan cells. The highly dynamic structures of IFs are important for the structural and mechanical integrity of the cell, but are involved in organization and signaling processes.[1,2] The assembly process of IFs differs fundamentally from the polymerization of microfilaments and microtubules. While the latter filaments polymerize from globular subunits by polar association,[3] the assembly process of IFs is assumed to consist of several hierarchical substeps. Vimentin occurs in cells of mesenchymal origin such as fibroblasts or blood

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