Generation of living cell arrays for atomic force microscopy studies.

Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.

Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy.

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety of adhesins that interact with host ligands. The spatial organization of these adhesins on the cellular interface is however poorly understood, mainly because of the lack of an instrument able to track single molecules on single cells. In this context, the atomic force microscope (AFM) makes it possible to analyze the force signature of single proteins on single cells. The present study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed that the adhesins at the cell surface were organized in nanodomains composed of free or aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids nanodomains will help in understanding the virulence factors of C. albicans.

A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts.

A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of ß-(1,3) and ß-(1,6)-glucan in the total ß-glucan of the cell wall. In the first step, chitin and ß-(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-ß-(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo-ß-(1,6)-glucanase from Trichoderma harzianum with ß-glucosidase from Aspergillus niger to hydrolyse the remaining ß-glucan. This latter component corresponded to a highly branched ß-(1,6)-glucan that contained about 75-80% of linear ß-(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of ß-(1,3), ß-(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of ß-(1,3)-glucan accompanied by an increase of ß-(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Uncovering by atomic force microscopy of an original circular structure at the yeast cell surface in response to heat shock.

BACKGROUND: Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h. RESULTS: We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2-3 µm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young's Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton. CONCLUSIONS: Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.

Atomic Force Microscopy and pharmacology: from microbiology to cancerology.

BACKGROUND: Atomic Force Microscopy (AFM) has been extensively used to study biological samples. Researchers take advantage of its ability to image living samples to increase our fundamental knowledge (biophysical properties/biochemical behavior) on living cell surface properties, at the nano-scale. SCOPE OF REVIEW: AFM, in the imaging modes, can probe cells morphological modifications induced by drugs. In the force spectroscopy mode, it is possible to follow the nanomechanical properties of a cell and to probe the mechanical modifications induced by drugs. AFM can be used to map single molecule distribution at the cell surface. We will focus on a collection of results aiming at evaluating the nano-scale effects of drugs, by AFM. Studies on yeast, bacteria and mammal cells will illustrate our discussion. Especially, we will show how AFM can help in getting a better understanding of drug mechanism of action. MAJOR CONCLUSIONS: This review demonstrates that AFM is a versatile tool, useful in pharmacology. In microbiology, it has been used to study the drugs fighting Candida albicans or Pseudomonas aeruginosa. The major conclusions are a better understanding of the microbes' cell wall and of the drugs mechanism of action. In cancerology, AFM has been used to explore the effects of cytotoxic drugs or as an innovative diagnostic technology. AFM has provided original results on cultured cells, cells extracted from patient and directly on patient biopsies. GENERAL SIGNIFICANCE: This review enhances the interest of AFM technologies for pharmacology. The applications reviewed range from microbiology to cancerology.

Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus.

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Use of atomic force microscopy (AFM) to explore cell wall properties and response to stress in the yeast Saccharomyces cerevisiae.

Over the past 20 years, the yeast cell wall has been thoroughly investigated by genetic and biochemical methods, leading to remarkable advances in the understanding of its biogenesis and molecular architecture as well as to the mechanisms by which this organelle is remodeled in response to environmental stresses. Being a dynamic structure that constitutes the frontier between the cell interior and its immediate surroundings, imaging cell surface, measuring mechanical properties of cell wall or probing cell surface proteins for localization or interaction with external biomolecules are among the most burning questions that biologists wished to address in order to better understand the structure-function relationships of yeast cell wall in adhesion, flocculation, aggregation, biofilm formation, interaction with antifungal drugs or toxins, as well as response to environmental stresses, such as temperature changes, osmotic pressure, shearing stress, etc. The atomic force microscopy (AFM) is nowadays the most qualified and developed technique that offers the possibilities to address these questions since it allows working directly on living cells to explore and manipulate cell surface properties at nanometer resolution and to analyze cell wall proteins at the single molecule level. In this minireview, we will summarize the most recent contributions made by AFM in the analysis of the biomechanical and biochemical properties of the yeast cell wall and illustrate the power of this tool to unravel unexpected effects caused by environmental stresses and antifungal agents on the surface of living yeast cells.

3D-fection: cell transfection within 3D scaffolds and hydrogels.

BACKGROUND: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

Dendrimer functionalization of gold surface improves the measurement of protein-DNA interactions by surface plasmon resonance imaging.

Surface Plasmon Resonance imaging (SPRi) is a label free technique typically used to follow biomolecular interactions in real time. SPRi offers the possibility to simultaneously investigate numerous interactions and is dedicated to high throughput analysis. However, precise determination of binding constants between partners is not highly reliable. We report here a dendrimer functionalization of gold surface that significantly improves selectivity of the detection of protein-DNA interactions. We showed that amino-gold surface functionalization with phosphorus dendrimers of fourth generation (G4) allowed complete coverage of the gold surface and the increase of the surface roughness. We optimized the conditions for DNA probe deposition to allow accurate detection of a well-known protein-DNA interaction involved in bacterial chromosome segregation. Using this G4-functionalized surface, the specificity of the SPRi response was significantly improved allowing discrimination between protein and DNA interactions of different strengths. Kinetic constants similar to those obtained with other techniques currently used in molecular biology were only obtained with the G4 dendrimer functionalized surface. This study demonstrated the benefit of using dendrimeric surfaces for sensitive high throughput SPRi analysis.

Nanoscale effects of antibiotics on P. aeruginosa.

Studying living bacteria at the nanoscale in their native liquid environment opens an unexplored landscape. We focus on Pseudomonas aeruginosa and demonstrate how the cell wall is biophysically affected at the nanoscale by two reference antibiotics (ticarcillin and tobramycin). The elasticity of the cells drops dramatically after treatment (from 263 ± 70 kPa to 50 ± 18 and 24 ± 4 kPa, respectively on ticarcillin- and tobramycin-treated bacteria) and major micro- and nano-morphological modifications are observed (the surface roughness of native, ticarcillin- and tobramycin-treated bacteria are respectively 2.5, 0.8, and 4.4 nm for a surface area of 40,000 nm²). Thus the nanoscale approach in liquid is valid and can be extended. FROM THE CLINICAL EDITOR: Pseudomonas aeruginosa cell wall was demonstrated to be biophysically affected at the nanoscale by two reference antibiotics, ticarcillin, and tobramycin, with the elasticity dropping dramatically after treatment.