Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Mechanistic Understanding of Protein Corona Formation around Nanoparticles: Old Puzzles and New Insights.

Although a wide variety of nanoparticles (NPs) have been engineered for use as disease markers or drug delivery agents, the number of nanomedicines in clinical use has hitherto remained small. A key obstacle in nanomedicine development is the lack of a deep mechanistic understanding of NP interactions in the bio-environment. Here, the focus is on the biomolecular adsorption layer (protein corona), which quickly enshrouds a pristine NP exposed to a biofluid and modifies the way the NP interacts with the bio-environment. After a brief introduction of NPs for nanomedicine, proteins, and their mutual interactions, research aimed at addressing fundamental properties of the protein corona, specifically its mono-/multilayer structure, reversibility and irreversibility, time dependence, as well as its role in NP agglomeration, is critically reviewed. It becomes quite evident that the knowledge of the protein corona is still fragmented, and conflicting results on fundamental issues call for further mechanistic studies. The article concludes with a discussion of future research directions that should be taken to advance the understanding of the protein corona around NPs. This knowledge will provide NP developers with the predictive power to account for these interactions in the design of efficacious nanomedicines.

Pax6 organizes the anterior eye segment by guiding two distinct neural crest waves.

Cranial neural crest (NC) contributes to the developing vertebrate eye. By multidimensional, quantitative imaging, we traced the origin of the ocular NC cells to two distinct NC populations that differ in the maintenance of sox10 expression, Wnt signalling, origin, route, mode and destination of migration. The first NC population migrates to the proximal and the second NC cell group populates the distal (anterior) part of the eye. By analysing zebrafish pax6a/b compound mutants presenting anterior segment dysgenesis, we demonstrate that Pax6a/b guide the two NC populations to distinct proximodistal locations. We further provide evidence that the lens whose formation is pax6a/b-dependent and lens-derived TGFß signals contribute to the building of the anterior segment. Taken together, our results reveal multiple roles of Pax6a/b in the control of NC cells during development of the anterior segment.

Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51.

Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV-visible absorption spectroscopy. In both enzymes, the P450 → P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 → P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme-thiolate ligand.

RNA polymerase II clusters form in line with surface condensation on regulatory chromatin.

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid-liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super-resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid-phase condensation at concentrations that are too low for canonical liquid-liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface-associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding.

Protein adsorption onto nanomaterials engineered for theranostic applications.

The key role of biomolecule adsorption onto engineered nanomaterials for therapeutic and diagnostic purposes has been well recognized by the nanobiotechnology community, and our mechanistic understanding of nano-bio interactions has greatly advanced over the past decades. Attention has recently shifted to gaining active control of nano-bio interactions, so as to enhance the efficacy of nanomaterials in biomedical applications. In this review, we summarize progress in this field and outline directions for future development. First, we briefly review fundamental knowledge about the intricate interactions between proteins and nanomaterials, as unraveled by a large number of mechanistic studies. Then, we give a systematic overview of the ways that protein-nanomaterial interactions have been exploited in biomedical applications, including the control of protein adsorption for enhancing the targeting efficiency of nanomedicines, the design of specific protein adsorption layers on the surfaces of nanomaterials for use as drug carriers, and the development of novel nanoparticle array-based sensors based on nano-bio interactions. We will focus on particularly relevant and recent examples within these areas. Finally, we conclude this topical review with an outlook on future developments in this fascinating research field.

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures.

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.

Exploring the energy landscape of a SAM-I riboswitch.

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-L-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated 'hub' state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

Formation of a Monolayer Protein Corona around Polystyrene Nanoparticles and Implications for Nanoparticle Agglomeration.

Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so-called "protein corona." An in-depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl-functionalized (PS-COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS-OSO3 H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein-NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS-OSO3 H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

Simulation of FRET dyes allows quantitative comparison against experimental data.

Fully understanding biomolecular function requires detailed insight into the systems' structural dynamics. Powerful experimental techniques such as single molecule Förster Resonance Energy Transfer (FRET) provide access to such dynamic information yet have to be carefully interpreted. Molecular simulations can complement these experiments but typically face limits in accessing slow time scales and large or unstructured systems. Here, we introduce a coarse-grained simulation technique that tackles these challenges. While requiring only few parameters, we maintain full protein flexibility and include all heavy atoms of proteins, linkers, and dyes. We are able to sufficiently reduce computational demands to simulate large or heterogeneous structural dynamics and ensembles on slow time scales found in, e.g., protein folding. The simulations allow for calculating FRET efficiencies which quantitatively agree with experimentally determined values. By providing atomically resolved trajectories, this work supports the planning and microscopic interpretation of experiments. Overall, these results highlight how simulations and experiments can complement each other leading to new insights into biomolecular dynamics and function.

Assessment of in vitro particle dosimetry models at the single cell and particle level by scanning electron microscopy.

BACKGROUND: Particokinetic models are important to predict the effective cellular dose, which is key to understanding the interactions of particles with biological systems. For the reliable establishment of dose-response curves in, e.g., the field of pharmacology and toxicology, mostly the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) and Distorted Grid (DG) models have been employed. Here, we used high resolution scanning electron microscopy to quantify deposited numbers of particles on cellular and intercellular surfaces and compare experimental findings with results predicted by the ISDD and DG models. RESULTS: Exposure of human lung epithelial A549 cells to various concentrations of differently sized silica particles (100, 200 and 500 nm) revealed a remarkably higher dose deposited on intercellular regions compared to cellular surfaces. The ISDD and DG models correctly predicted the areal densities of particles in the intercellular space when a high adsorption ("stickiness") to the surface was emulated. In contrast, the lower dose on cells was accurately inferred by the DG model in the case of "non-sticky" boundary conditions. Finally, the presence of cells seemed to enhance particle deposition, as aerial densities on cell-free substrates were clearly reduced. CONCLUSIONS: Our results further validate the use of particokinetic models but also demonstrate their limitations, specifically, with respect to the spatial distribution of particles on heterogeneous surfaces. Consideration of surface properties with respect to adhesion and desorption should advance modelling approaches to ultimately predict the cellular dose with higher precision.

Supramolecular Self-Assembly Bioinspired Synthesis of Luminescent Gold Nanocluster-Embedded Peptide Nanofibers for Temperature Sensing and Cellular Imaging.

Metal nanoclusters (NCs) hold great potential as novel luminescent nanomaterials in many applications, while the synthesis of highly luminescent metal NCs still remains challenging. In this work, we report self-assembling peptides as a novel bioinspired scaffold capable of significantly enhancing the luminescence efficiency of gold nanoclusters (AuNCs). The resulting AuNCs capped with motif-designed peptides can self-assemble to form nanofiber structures, in which the luminescence of AuNCs is enhanced nearly 70-fold, with 21.3% quantum yield. The underlying mechanism responsible for the luminescence enhancement has been thoroughly investigated by the combined use of different spectroscopic and microscopic techniques. The resultant highly luminescent AuNC-decorated peptide nanofibers exhibit physicochemical properties that are advantageous for biological applications. As a proof of concept, we demonstrate the use of these nanostructure as fluorescent thermometers and for imaging living cells, both showing very promising results.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

In Situ Monitoring of the Intracellular Stability of Nanoparticles by Using Fluorescence Lifetime Imaging.

FLIMaging nanoparticle degradation: semiconductor and metal nanoparticle degradation has been observed in live cells over 3 d via the change of the characteristic luminescence lifetime using fluorescence lifetime imaging microscopy (FLIM). Thus, FLIM is a simple yet robust tool to examine the intracellular stability of photoluminescent nanoparticles in live cells, tissues, and organisms.

The Nature of a Hard Protein Corona Forming on Quantum Dots Exposed to Human Blood Serum.

Biological responses of cells and organisms to nanoparticle exposure crucially depend on the properties of the protein adsorption layer ("protein corona") forming on nanoparticle surfaces and their characterization is a crucial step toward a deep, mechanistic understanding of their build-up. Previously, adsorption of one type of model protein on nanoparticles was systematically studied in situ by using fluorescence correlation spectroscopy. Here, the first such study of interactions is presented between water-solubilized CdSe/ZnS quantum dots (QDs) and a complex biofluid, human blood serum. Despite the large number of proteins in serum, a protein layer of well-defined (average) thickness forming on QD surfaces is observed. Both the thickness and the apparent binding affinity depend on the type of QD surface ligand. Kinetic experiments reveal that the protein corona formed from serum is irreversibly bound, whereas the one formed from human serum albumin was earlier observed to be reversible. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, the most abundant serum proteins contributing to the formation of a hard corona on the QDs are identified.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Localization and dynamics of glucocorticoid receptor at the plasma membrane of activated mast cells.

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Engineered nanoparticles interacting with cells: size matters.

With the rapid advancement of nanoscience and nanotechnology, detailed knowledge of interactions between engineered nanomaterials and cells, tissues and organisms has become increasingly important, especially in regard to possible hazards to human health. This review intends to give an overview of current research on nano-bio interactions, with a focus on the effects of NP size on their interactions with live cells. We summarize common techniques to characterize NP size, highlight recent work on the impact of NP size on active and passive cellular internalization and intracellular localization. Cytotoxic effects are also discussed.

Caveolae disassemble upon membrane lesioning and foster cell survival.

Repair of lesions in the plasma membrane is key to sustaining cellular homeostasis. Cells maintain cytoplasmic as well as membrane-bound stores of repair proteins that can rapidly precipitate at the site of membrane lesions. However, little is known about the origins of lipids and proteins for resealing and repair of the plasma membrane. Here we study the dynamics of caveolar proteins after laser-induced lesioning of plasma membranes of mammalian C2C12 tissue culture cells and muscle cells of intact zebrafish embryos. Single-molecule diffusivity measurements indicate that caveolar clusters break up into smaller entities after wounding. Unlike Annexins and Dysferlin, caveolar proteins do not accumulate at the lesion patch. In caveolae-depleted cavin1a knockout zebrafish embryos, lesion patch formation is impaired, and injured cells show reduced survival. Our data suggest that caveolae disassembly releases surplus plasma membrane near the lesion to facilitate membrane repair after initial patch formation for emergency sealing.