Fania Geiger

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far‐field methods have been developed allowing a substantial increase of resolution (“superresolution microscopy”, or “lightoptical nanoscopy”). This opens an avenue to ‘nano‐image’ intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDMPhymod. Generally, this SPDM method is based on high‐precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual‐color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20‐nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition

Summary The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine–aspartic acid (KD)x motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA4H4) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD)x charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved.

Dual Functionalization of Rod-Shaped Viruses on Single Coat Protein Subunits

Plant viruses are emerging as versatile tools for nanotechnology applications since it is possible to modify their multivalent protein surfaces and thereby introduce and display new functionalities. In this chapter, we describe a tobacco mosaic virus (TMV) variant that exposes two selectively addressable amino acid moieties on each of its 2130 coat protein (CP) subunits. A lysine as well as a cysteine introduced at accessible sites of every CP can be modified with amino- and/or thiol-reactive chemistry such as N-hydroxysuccinimide esters (NHS ester) and maleimide containing reagents alone or simultaneously. This enables the pairwise immobilization of distinct molecules in close vicinity to each other on the TMV surface by simple standard conjugation protocols. We describe the generation of the mutations, the virus propagation and isolation as well as the dual functionalization of the TMV variant with two fluorescent dyes. The labeling is evaluated by SDS-PAGE and spectrophotometry and the degree of labeling (DOL) calculated.

Laminin-521 promotes quiescence in isolated stellate cells from rat liver

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.

Correction: Hyaluronsäuregele zur Druckregulierung in der Glaukomtherapie

Correction: Ophthalmologe 2017 https://doi.org/10.1007/s00347-017-0602-z Sehr geehrter Leser, sehr geehrte Leserin, leider wurde der oben genannte Beitrag online zunachst mit fehlerhaftem deutschen Titel veroffentlicht. Wir bitten, den korrekten Titel Hyaluronsauregele zur Druckregulierung in der Glaukomtherapie zu berucksichtigen und den Fehler zu entschuldigen. Die RedaktionCorrection:Ophthalmologe 2017 https://doi.org/10.1007/s00347-017-0602-z Sehr geehrter Leser,sehr geehrte Leserin,leider wurde der oben genannte Beitrag online zunächst mit fehlerhaftem deutschen Titel veröffentlicht. Wir bitten, den korrekten Titel Hyaluronsäuregele zur Druckregulierung in der Glaukomtherapie zu berücksichtigen und den Fehler zu entschuldigen.Die Redaktion