Super-Resolved Protein Imaging Using Bifunctional Light-Up Aptamers.

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers.

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.

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.

Stimulated emission double depletion nanoscopy with background correction at the single-pixel level.

Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific background arises from incomplete depletion and re-excitation by the depletion beam. Various approaches have been proposed to remove the background from a STED image, some of which rely on the acquisition of a separate background image that is subtracted from the STED image with a weighting factor.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form.

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer.

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.

Genetically encodable fluorescent protein markers in advanced optical imaging.

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene.

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

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.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway.

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer.

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.

Two plus one is almost three: a fast approximation for multi-view deconvolution.

Multi-view deconvolution is a powerful image-processing tool for light sheet fluorescence microscopy, providing isotropic resolution and enhancing the image content. However, performing these calculations on large datasets is computationally demanding and time-consuming even on high-end workstations. Especially in long-time measurements on developing animals, huge amounts of image data are acquired.

Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells.

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood.

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 Mg 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.

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.

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy.

Optical fluorescence microscopy has taken center stage in the exploration of biological structure and dynamics, especially on live specimens, and super-resolution imaging methods continue to deliver exciting new insights into the molecular foundations of life. Progress in the field, however, crucially hinges on advances in fluorescent marker technology. Among these, fluorescent proteins (FPs) of the GFP family are advantageous because they are genetically encodable, so that live cells, tissues or organisms can produce these markers all by themselves.

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).

Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy.

Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is widely used, but it faces limitations due to (1) the heterogeneity of biomembranes on the nanoscale and (2) the intrinsic motion of membranes with respect to the focus. Here we introduce a new confocal microscopy-based fluctuation spectroscopy technique aimed at alleviating these two problems, called axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS).

Wavelet-based background and noise subtraction for fluorescence microscopy images.

Fluorescence microscopy images are inevitably contaminated by background intensity contributions. Fluorescence from out-of-focus planes and scattered light are important sources of slowly varying, low spatial frequency background, whereas background varying from pixel to pixel (high frequency noise) is introduced by the detection system. Here we present a powerful, easy-to-use software, wavelet-based background and noise subtraction (WBNS), which effectively removes both of these components.

Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer.

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties.

Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

Brownian motion-based nanoparticle sizing-A powerful approach for in situ analysis of nanoparticle-protein interactions.

A key hurdle toward effective application of nanoparticles (NPs) in biomedicine is still the incomplete understanding of the biomolecular adsorption layer, the so-called protein corona, which inevitably forms around NPs when they are immersed in a biofluid. NP sizing techniques via the analysis of Brownian motions offer a powerful way to measure the thickness of the protein corona in situ. Here, the fundamentals of three techniques, dynamic light scattering, fluorescence correlation spectroscopy, and nanoparticle tracking analysis are briefly summarized.

MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly.

The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects.

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.