Katja Henzler

Adsorption of β-Lactoglobulin on Spherical Polyelectrolyte Brushes: Direct Proof of Counterion Release by Isothermal Titration Calorimetry

The thermodynamics and the driving forces of the adsorption of beta-lactoglobulin on spherical polyelectrolyte brushes (SPB) are investigated by isothermal titration calorimetry (ITC). The SPB consist of a polystyrene core onto which long chains of poly(styrene sulfonate) are grafted. Adsorption isotherms are obtained from measurements by ITC. The analysis by ITC shows clearly that the adsorption process is solely driven by entropy while DeltaH > 0. This finding is in accordance with the proposed mechanism of counterion release: Patches of positive charges on the surface of the proteins become multivalent counterions of the polyelectrolyte chains, thereby releasing the counterions of the protein and the polyelectrolyte. A simple statistical-mechanical model fully corroborates the proposed mechanism. The present analysis shows clearly the fundamental importance of counterion release for protein adsorption on charged interfaces and charged polymeric layers.

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane

Summary Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM).

Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities.

Towards nanoscale composite particles of dual complexity

The fabrication of heteroaggregates comprising inorganic and organic nanoparticles of different sizes is reported. Control over the assembly of nanoscale functional building units is of great significance to many practical applications. Joining together different spherical nanoparticles in a defined manner allows control over the shape of the composites. If two types of constituents are chosen that differ in size, the surfaces of the composites exhibit two specific radii of curvature, yielding aggregates of dual surface roughness. Moreover, if the constituents consist of different materials, the resulting heteroaggregates feature both compositional and interfacial anisotropy, offering unprecedented perspectives for custom-tailored colloids. This study describes a two-step approach towards such designer particles. At first, amine-modified polystyrene particles with 154 nm diameter were assembled into clusters of well-defined configurations. Onto these, oppositely charged inorganic particles with diameters of only a few nanometres were deposited by direct uptake from solution, resulting in numerous functional entities all over the surface of the polymer clusters. Despite the fact that oppositely charged constituents are brought together, charge reversal by uptake of nanoparticles allows for stable suspensions of heterocomposites. Hence, the possibility to assemble particles into nanoscale heterocomposites with full control over shape, composition, and surface roughness is demonstrated.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells.

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness.

New Insight into Microgel-Stabilized Emulsions Using Transmission X-ray Microscopy: Nonuniform Deformation and Arrangement of Microgels at Liquid Interfaces

Microgel-covered interfaces, e.g., in emulsions, have attracted much interest lately. Different imaging techniques have been used to image these interfaces, either flat or curved, to investigate their properties and appearance. Techniques such as cryogenic scanning electron microscopy (cryo-SEM) and confocal microscopy have provided valuable insight into microgel-covered systems but still have some disadvantages such as part of the microgels being trapped in vitrified liquid or the need for fluorescent markers. Some of these disadvantages can be overcome by using transmission X-ray microscopy (TXM), which has the advantage of allowing the investigation of adsorbed and free microgels simultaneously. We used TXM to acquire tomographic image series of microgel-covered droplets and calculated 3D reconstructions from these image stacks. As a result, we could show that microgels deform anisotropically and penetrate the oil droplets in the hydrated state. Additionally, 3D reconstruction gives an idea of the arrangement of microgels adsorbed to oil droplets and reveals that droplet stabilization is possible without full coverage of the interface with polymer segments.

Thermosensitive Cu2O–PNIPAM core–shell nanoreactors with tunable photocatalytic activity

We report a facile and novel method for the fabrication of Cu2O@PNIPAM core-shell nanoreactors using Cu2O nanocubes as the core. The PNIPAM shell not only effectively protects the Cu2O nanocubes from oxidation, but also improves the colloidal stability of the system. The Cu2O@PNIPAM core-shell microgels can work efficiently as photocatalyst for the decomposition of methyl orange under visible light. A significant enhancement in the catalytic activity has been observed for the core-shell microgels compared with the pure Cu2O nanocubes. Most importantly, the photocatalytic activity of the Cu2O nanocubes can be further tuned by the thermosensitive PNIPAM shell, as rationalized by our recent theory.

Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-ray Scattering

The immobilization of bovine serum albumins (BSA) onto cationic spherical polyelectrolyte brushes (SPB) consisting of a solid polystyrene (PS) core and a densely grafted poly(2-aminoethyl methacrylate hydrochloride) (PAEMH) shell was studied by small-angle X-ray scattering (SAXS). The observed dynamics of adsorption of BSA onto SPB by time-resolved SAXS can be divided into two stages. In the first stage (tens of milliseconds), the added proteins as in-between bridge instantaneously caused the aggregation of SPB. Then BSA penetrated into the brush layer driven by electrostatic attractions, and reached equilibrium in the second stage (tens of seconds). The amount of BSA immobilized onto brush layer reached the maximum when pH was increased to about 6.1 and BSA concentration to 10 g/L. The cationic SPB were confirmed to provide stronger adsorption capacity for BSA compared to anionic ones.

Electronic structure of individual hybrid colloid particles studied by near-edge X-ray absorption fine structure (NEXAFS) spectroscopy in the X-ray microscope.

The electronic structure of individual hybrid particles was studied by nanoscale near-edge X-ray absorption spectromicroscopy. The colloidal particles consist of a solid polystyrene core and a cross-linked poly-N-(isopropylacrylamide) shell with embedded crystalline titanium dioxide (TiO(2)) nanoparticles (d = 6 ± 3 nm). The TiO(2) particles are generated in the carrier network by a sol-gel process at room temperature. The hybrid particles were imaged with photon energy steps of 0.1 eV in their hydrated environment with a cryo transmission X-ray microscope (TXM) at the Ti L(2,3)-edge. By analyzing the image stacks, the obtained near-edge X-ray absorption fine structure (NEXAFS) spectra of our individual hybrid particles show clearly that our synthesis generates TiO(2) in the anastase phase. Additionally, our spectromicroscopy method permits the determination of the density distribution of TiO(2) in single carrier particles. Therefore, NEXAFS spectroscopy combined with TXM presents a unique method to get in-depth insight into the electronic structure of hybrid materials.

Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS-TXM imaging

In order to take full advantage of novel functional materials in the next generation of sensorial devices scalable processes for their fabrication and utilization are of great importance. Also understanding the processes lending the properties to those materials is essential. Among the most sought-after sensor applications are low-cost, highly sensitive and selective metal oxide based gas sensors. Yet, the surface reactions responsible for provoking a change in the electrical behavior of gas sensitive layers are insufficiently comprehended. Here, we have used near-edge x-ray absorption fine structure spectroscopy in combination with x-ray microscopy (NEXAFS-TXM) for ex-situ measurements, in order to reveal the hydrogen sulfide induced processes at the surface of copper oxide nanoparticles, which are ultimately responsible for triggering a percolation phase transition. For the first time these measurements allow the imaging of trace gas induced reactions and the effect they have on the chemical composition of the metal oxide surface and bulk. This makes the new technique suitable for elucidating adsorption processes in-situ and under real operating conditions.