Single-Molecule Atomic Force Microscopy Reveals Clustering of the Yeast Plasma-Membrane Sensor Wsc1

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches

Calcium Ions Promote Formation of Amyloid β-Peptide (1–40) Oligomers Causally Implicated in Neuronal Toxicity of Alzheimer's Disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1–40) and that of Aβ(1–40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca2+, Aβ(1–40) preferentially formed oligomers similar to those formed by Aβ(1–40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1–40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1–40), that have been implicated in the pathogenesis of AD

Surfaces hétérogènes contrôlées et systèmes diphasiques bidimensionnels

This thesis presents the elaboration and the characterisation of controlled heterogeneous surfaces in order to study the effect of the heterogeneities on the wetting properties. These surfaces are obtained by preparing Langmuir-Blodgett films with a mixture of two amphiphilic molecules. These molecules were chosen for their different wetting properties : the first one is a long chain fatty acid while the second one is a partially fluorinated surfactant. The mixed films are first characterised at the air-water interface (Langmuir monolayers). In order to determine the mixing behaviour of the two molecules, the mixed films were studied at different scales using several experimental techniques. The results show that a complete phase separation takes place over the whole range of molar fractions and whatever the surface pressure. Circular domains of the fatty acid are evidenced.The mixed films are then transferred on solid substrate. In order to obtain well anchored films, the transfer parameters were adapted. The molecular arrangement in the films is determined using grazing incidence X-ray diffraction with two angles of incidence of the beam to change its penetration depth. We also used atomic force microscopy to obtain surface topography and to check on the absence of defects. In the same way as at the air-water interface, circular domains are visualised. These domains coexist with a continuous phase which presents a labyrinthine structure attributed to a molecular reorganisation during transfer.Then, we obtain a system that appears well adapted to study the wetting properties of heterogeneous surfaces : the two molecules are completely phase-separated and each phase has different properties.Cette thèse présente un travail d?élaboration et de caractérisation de surfaces hétérogènes contrôlées afin d?étudier l?effet des hétérogénéités sur les propriétés de mouillage. Ces surfaces sont obtenues par la technique de Langmuir-Blodgett à partir du mélange de deux molécules amphiphiles différentes. Ces molécules ont été choisies pour leurs propriétés de mouillage différentes : la première est un acide gras à longue chaîne tandis que la seconde est une molécule à chaîne hydrophobe partiellement fluorée.Dans un premier temps, les films mixtes sont caractérisés à l?interface eau-air (films de Langmuir). Afin de déterminer la nature des interactions entre les constituants, les films mixtes ont été étudiés à différentes échelles en utilisant différentes techniques expérimentales. Les résultats montrent que le système présente une séparation de phase complète quelle que soit la fraction molaire de l?un des constituants et dès les basses pressions de surface. Des domaines circulaires d?acide sont mis en évidence.Les films sont ensuite transférés sur substrat solide. Pour obtenir un bon ancrage des films, les paramètres de transfert ont été adaptés. L?arrangement moléculaire au sein des films a été déterminé de façon précise par diffraction X sous incidence rasante, notamment par variation de l?angle d?incidence des photons sur l?échantillon de façon à pénétrer plus ou moins dans le film. Les films ont également été imagés par microscopie à force atomique, ce qui permet de déterminer l?état des surfaces et notamment l?absence de défauts. De la même façon qu?à la surface de l?eau, des domaines circulaires d?acide sont observés. Ces domaines coexistent avec une phase continue qui présente une structure labyrinthique attribuée à une réorganisation moléculaire lors du transfert.Nous avons ainsi obtenu un système bien adapté à l?étude des propriétés de mouillage de surfaces inhomogènes puisqu?il présente deux phases séparées possédant chacune des propriétés différentes

Probing molecular recognition sites on biosurfaces using AFM.

Knowledge of the molecular forces that drive receptor-ligand interactions is a key to gain a detailed understanding of cell adhesion events and to develop novel applications in biomaterials science. Until recently, there was no tool available for analyzing and mapping these forces on complex biosurfaces like cell surfaces. During the past decade, however, single-molecule atomic force microscopy (AFM) has opened exciting new opportunities for detecting and localizing molecular recognition forces on artificial biosurfaces and on living cells. In this review, we describe the general principles of the AFM technique, present procedures commonly used to prepare samples and tips, and discuss a number of applications that are relevant to the field of biomaterials

Atomic force microscopy demonstrates that disulfide bridges are required for clustering of the yeast cell wall integrity sensor wsc1.

In yeasts, cell surface stresses are detected by a family of plasma membrane sensors. Among these, Wsc1 contains an extracellular cysteine-rich domain (CRD), which mediates sensor clustering and is believed to anchor the sensor in the cell wall. Although the formation of Wsc1 clusters and their interaction with the intracellular pathway components are important for proper stress signaling, the molecular mechanisms underlying clustering remain poorly understood. Here, we used the combination of single-molecule atomic force microscopy (AFM) with genetic manipulations to demonstrate that Wsc1 clustering involves disulfide bridges of the CRD. Using AFM tips carrying nitrilotriacetate groups, we mapped the distribution of individual His-tagged sensors on living yeast cells. While Wsc1 formed nanoscale clusters on native cells, clustering was no longer observed after treatment with the reducing agent dithiothreitol (DTT), indicating that intra- or intermolecular disulfide bridges are required for clustering. Moreover, DTT treatment resulted in a significant increase in cell surface roughness, suggesting that disulfide bridges between other cell-wall proteins are crucial for proper cell surface topology. The remarkable sensor properties unravelled here may well apply to other sensors and receptors with cysteine-rich domains throughout biology. Our combined method of AFM with genetic manipulations offers great prospects to explore the mechanisms underlying the clustering of cell surface proteins

Measuring Cell Wall Thickness in Living Yeast Cells Using Single Molecular Rulers.

Traditionally, the structural details of microbial cell walls are studied by thin-section electron microscopy, a technique that is very demanding and requires vacuum conditions, thus precluding live cell experiments. Here, we present a method integrating single-molecule atomic force microscopy (AFM) and protein design to measure cell wall thickness in a living yeast cell. The basic idea relies on the expression of His-tagged membrane sensors of increasing lengths in yeast and their subsequent specific detection at the cell surface using a modified AFM tip. After establishing the method on a wild-type strain, we demonstrate its potential by measuring changes in cell wall thickness within a few nanometers range, which result from (bio)chemical treatments or from mutations affecting the cell wall structure. The single molecular ruler method presented here not only avoids cell fixation artifacts but also provides new opportunities for studying the dynamics of microbial cell walls during growth, drug action, or enzymatic modification

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy.

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities

Atomic force microscopy: A powerful tool for studying bacterial swarming motility

Swarmingmotility is a fascinating phenomenon by which some bacteria use flagella to move over solid surfaces. Understanding the molecular mechanisms underlying swarmingmotility requires studying the factors that induce and control flagella expression in swarming cells. Traditionally, flagella are observed by optical or electron microscopy, but none of these techniques combine versatility and easiness, with quantitative and high-resolution information. We report an atomicforce microscopy (AFM)-based approach for the fast imaging of bacterial phenotypes (cell shape, flagella expression) in swarmingmotility studies. Cells from the Gram-positive bacterium Bacillus thuringiensis sv. israelensis were inoculated on energy-rich media containing increasing agar concentrations. Following swarming assays (2 days), the cell morphology and the amount of flagella were directly observed by AFM imaging in air. Consistent with the macroscopic swarming behavior, cells harvested from the rim of colonies spreading on soft agar were hyperflagellated, elongated and arranged in chains. Increasing the agar concentration led to much lower amounts of flagella and to shorter rod-shaped cells, a finding consistent with the slower swarmingmotility of the cells. Cells taken from colony centers on soft and hard agar surfaces were generally non-flagellated, rod-shaped, rarely arranged in chains, and exhibited lysis and sporulation. This study shows that AFM imaging can readily discriminate between swarming and non-swarming cells, and quantify their morphological details, thus offering an important tool to study the dynamics of bacterial populations

Microbial nanoscopy: a closer look at microbial cell surfaces.

How cell envelope constituents are spatially organised and how they interact with the environment are key questions in microbiology. Unlike other bioimaging tools, atomic force microscopy (AFM) provides information about the nanoscale surface architecture of living cells and about the localization and interactions of their individual constituents. These past years have witnessed remarkable advances in our use of the AFM molecular toolbox to observe and force probe microbial cells. Recent milestones include the real-time imaging of the nanoscale organization of cell walls, the quantification of subcellular chemical heterogeneities, the mapping and functional analysis of individual cell wall constituents and the analysis of the mechanical properties of single receptors and sensors