Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

A Bayesian approach to tracking in single molecule fluorescence microscopy

Using fluorescence microscopy with single molecule sensitivity it is now possible to follow the movement of individual fluorophore tagged molecules such as proteins and lipids in the cell membrane with nanometer precision. These experiments are important as they allow many key biological processes on the cell membrane and in the cell, such as transcription, translation and DNA replication, to be studied at new levels of detail. Computerized microscopes generate sequences of images (in the order of tens to hundreds) of the molecules diffusing and one of the challenges is to track these molecules to obtain reliable statistics such as speed distributions, diffusion patterns, intracellular positioning, etc. The data set is challenging because the molecules are tagged with a single or small number of fluorophores, which makes it difficult to distinguish them from the background, the fluorophore bleaches irreversibly over time, the number of tagged molecules are unknown and there is occasional loss of signal from the tagged molecules. All these factors make accurate tracking over long trajectories difficult. Also the experiments are technically difficulty to conduct and thus there is a pressing need to develop better algorithms to extract the maximum information from the data. For this purpose we propose a Bayesian approach and apply our technique to synthetic and a real experimental data set.

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells. (C) 2008 Elsevier B.V. All rights reserved.

A robust algorithm for segmenting fluorescence images and its application to single-molecule counting

La microscopie par fluorescence de cellules vivantes produit de grandes quantités de données. Ces données sont composées d’une grande diversité au niveau de la forme des objets d’intérêts et possèdent un ratio signaux/bruit très bas. Pour concevoir un pipeline d’algorithmes efficaces en traitement d’image de microscopie par fluorescence, il est important d’avoir une segmentation robuste et fiable étant donné que celle-ci constitue l’étape initiale du traitement d’image. Dans ce mémoire, je présente MinSeg, un algorithme de segmentation d’image de microscopie par fluorescence qui fait peu d’assomptions sur l’image et utilise des propriétés statistiques pour distinguer le signal par rapport au bruit. MinSeg ne fait pas d’assomption sur la taille ou la forme des objets contenus dans l’image. Par ce fait, il est donc applicable sur une grande variété d’images. Je présente aussi une suite d’algorithmes pour la quantification de petits complexes dans des expériences de microscopie par fluorescence de molécules simples utilisant l’algorithme de segmentation MinSeg. Cette suite d’algorithmes a été utilisée pour la quantification d’une protéine nommée CENP-A qui est une variante de l’histone H3. Par cette technique, nous avons trouvé que CENP-A est principalement présente sous forme de dimère.

Dissection du processus d’export des ARNm nucléaires par des approches de molécules uniques chez Saccharomyces cerevisae

Enfermer le porteur de l’information génétique dans le noyau a obligée la cellule a créé un système de transport complexe, qui permet l’export d’un ARNm du noyau au cytoplasme. Le mécanisme général de l’export des ARNm est encore mal connu, même si les facteurs principaux ont été découverts il y a longtemps. De récents progrès en microscopie nous ont permis d’étudier directement le comportement des ARNm durant le processus d’export. Durant ma maitrise, nous avons été capables de localiser et suivre des ARNm en temps réel pour la première fois chez Saccharomyces cerevisiae. Nous avons créé un gène rapporteur en mettant le gène GLT1 sous le contrôle du promoteur GAL1. Nous avons aussi marqué l’ARNm de GLT1 avec plusieurs boucles PP7. L’ARNm sera visible après l’attachement de plusieurs protéines PP7-GFP aux boucles. En utilisant la technique d’imagerie en cellules vivantes, nous sommes capable de visualiser et suivre chaque ARNm, depuis son relâchement du site de transcription jusqu’à l’export. Une fois relâché du site de transcription, l’ARNm diffuse librement dans le nucléoplasme, mais une fois à la périphérie nucléaire, il commence à « scanner » l’enveloppe nucléaire avant d’être exporté. Nous avons trouvé que le « scanning » dépend de la présence des Myosin Like Proteins (Mlp1p et Mlp2p), protéines qui forment le panier nucléaire, car suite à la délétion de MLP1 et MLP2, les ARNm n’étaient plus capable de « scanner ». Nous avons également trouvé que la partie C-terminale de Mlp1p était nécessaire au « scanning ». De plus, suite à la délétion du gène TOM1, gène codant pour une ubiquitine ligase, les ARNm ont un comportement similaire aux ARNm d’une souche ∆mlp1/mlp2, suggérant que le « scanning » permet à Tom1p d’ubiquitiner Yra1p, ce qui causera son relâchement de l’ARNm. Également, nous avons montré que les ARNm endogènes MDN1 et CBL2 scannent aussi la périphérie nucléaire. Ensemble, nos résultats suggèrent que le scanning est un processus par lequel passent tout les ARNm nucléaire lorsqu’ils se retrouvent à la périphérie du noyau, pour initier plusieurs étapes de réarrangements nécessaires à leurs export. De plus, nous avons examiné le rôle de Yhr127p, une protéine nouvellement identifiée qui se lie à l’ARN. Après avoir marqué cette protéine avec la GFP, nous avons montré qu’elle forme des foci dans le noyau et que ces derniers vont disparaitre suite à l’arrêt de la transcription. La délétion de YHR127 à conduit à une augmentation de la transcription de quelques gènes spécifiques, mais n’affecte pas la capacité de la cellule à exporter les ARNm. Nos résultats suggèrent que cette protéine joue un rôle dans la régulation de la transcription et/ou dans la stabilité de l’ARNm.

Single molecule microscopy in 3D cell cultures and tissues

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

Clean localization super-resolution microscopy for 3D biological imaging

We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells. (C) 2016 Author(s).

Single-molecule dynamic triangulation

A proposal for using single molecules as nanoprobes capable of detecting the trajectory of an elementary charge is discussed in detail. Presented numerical simulations prove that this singlemolecule technique allows determination of a three-dimensional single-electron displacement within a few seconds with an accurocy better than 0.006 nm. Surprisingly, this significantly exceeds the accuracy with which the probe;, molecule itself can be localized (given the same measuring time by means of single-molecule microscopy. It is also shown that the optimal concentration of probe molecules in the vicinity of:the electron (i.e. the concentration which provides the best accuracy of the inferred electron displacement) is of the order of 10(-5) m.

Inferring diffusion in single live cells at the single-molecule level

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single-molecule and single-cell level can add significant insight into understanding molecular architectures of diffus- ing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell mem- brane under relatively physiological conditions compared with competing single-molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the mol- ecular level owing to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles; however, this generally requires more data points than is typical for single FP tracks owing to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes to dis-criminate multiple complex modes probabilistically. It is a computational approach that biologists can use to understand single-molecule features in live cells.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.