Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells

Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules

Rickettsiae Induce Microvascular Hyperpermeability via Phosphorylation of VE-Cadherins: Evidence from Atomic Force Microscopy and Biochemical Studies

The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein–protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses

Morphological and Structural Aspects of the Extremely Halophilic Archaeon Haloquadratum walsbyi

Ultrathin square cell Haloquadratum walsbyi from the Archaea domain are the most abundant microorganisms in the hypersaline water of coastal salterns and continental salt lakes. In this work, we explore the cell surface of these microorganisms using amplitude-modulation atomic-force microscopy in nearly physiological conditions. We demonstrate the presence of a regular corrugation with a periodicity of 16–20 nm attributed to the surface layer (S-layer) protein lattice, striped domains asymmetrically distributed on the cell faces and peculiar bulges correlated with the presence of intracellular granules. Besides, subsequent images of cell evolution during the drying process indicate the presence of an external capsule that might correspond to the giant protein halomucin, predicted by the genome but never before observed by other microscopy studies

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations. (C) 2000 Elsevier Science B.V. All rights reserved

Nanoscale Modification Of Supported Lipid Membranes: Synergetic Effect Of Phospholipase D And Viral Fusion Peptides

peer reviewedUnderstanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. In this study, we used atomic force microscopy (AFM) to address the crucial question as to whether negatively curved lipids influence the ability of a viral fusion peptide to perturb the organization of supported lipid bilayers. To this end, an original approach was developed that makes use of an AFM tip functionalized with phospholipase D (PLD) enzymes to generate in situ small amounts of negatively curved phosphatidic acid (PA) in mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers. Real-time AFM imaging revealed that this nanomodification dramatically enhanced subsequent interaction with the simian immunodeficiency virus (SIV) fusion peptide. At short incubation time, the SIV peptide induced a 1.9 nm thickness reduction of the DPPC domains, reflecting either interdigitation or fluidification of the lipids. At longer incubation time, these depressed domains transformed into elevated striated domains, protruding one to several nanometers above the bilayer surface. Two complementary experiments, i.e. addition of the peptide onto DOPC/DPPC/DOPA bilayers or onto DOPC/DPPC bilayers pretreated with a PLD solution, confirmed that both PA and SIV peptides are required to induce depressed and striated domains. Accordingly, this is the first time that a high-resolution imaging technique is used to demonstrate that negatively curved lipids affect the membrane activity of fusion peptides. We believe the nanoscale approach presented here, i.e. use of enzyme-functionalized AFM tips to modify lipid bilayers, will find exciting new applications in nanobiotechnology for the design of biomimetic surfaces