50nm-scale localization of single unmodified, isotopically enriched, proteins in cells.

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when (14)N isotopes are replaced with (15)N. (15)N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS).

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor.

Background: There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear.

Presentation Of The Hypothesis: In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell.

Hybolites: novel therapeutic tools for targeting hyperstructures in bacteria.

The scarcity of new molecules that can act on bacteria is a major problem. New strategies for developing such molecules might be based on recent concepts in microbiology. Hyperstructures are large assemblies of molecules and macromolecules that perform functions such as DNA replication, RNA degradation and chemotaxis and the interactions between hyperstructures have been proposed to constitute an intermediate level of organisation in cells.

Chemical microscopy of biological samples by dynamic mode secondary ion mass spectrometry.

3D chemical microscopy is one of the emerging applications of secondary ion mass spectrometry (SIMS) in biology. Tissues, cells, extracellular matrices, and polymer films can be imaged at present with a lateral resolution of 50 nm and depth resolution of 1 nm using the latest generation of CAMECA magnetic sector NanoSIMS 50 or with a lower lateral resolution (above 100 nm) using IMS 4f Cameca SIMS equipped with cold stage. Dynamic mode SIMS analysis is performed in ultrahigh vacuum and thus requires specific and careful preparation of biological samples aimed at preserving and minimizing destruction of the original structural and chemical properties of the samples.

Method for macromolecular colocalization using atomic recombination in dynamic SIMS.

Localizing two or more components of assemblies in biological systems requires both continued development of fluorescence techniques and invention of entirely new techniques. Candidates for the latter include dynamic secondary ion mass spectrometry (D-SIMS). The latest generation of D-SIMS, the Cameca NanoSIMS 50, permits the localization of specific, isotopically labeled molecules and macromolecules in sections of biological material with a resolution in the tens of nanometers and with a sensitivity approaching in principle that of a single protein.

A stochastic automaton shows how enzyme assemblies may contribute to metabolic efficiency.

Background: The advantages of grouping enzymes into metabolons and into higher order structures have long been debated. To quantify these advantages, we have developed a stochastic automaton that allows experiments to be performed in a virtual bacterium with both a membrane and a cytoplasm. We have investigated the general case of transport and metabolism as inspired by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) for glucose importation and by glycolysis.

Steady-state kinetic behaviour of two- or n-enzyme systems made of free sequential enzymes involved in a metabolic pathway.

The overall rate of functioning of a set of free sequential enzymes of the Michaelis-Menten type involved in a metabolic pathway has been computed as a function of the concentration of the initial substrate under steady-state conditions. Curves monotonically increasing up to a saturation plateau have been obtained in all cases. The shape of these curves is sometimes, but not usually, close to that of a hyperbola.

Steady-state kinetic behaviour of functioning-dependent structures.

A fundamental problem in biochemistry is that of the nature of the coordination between and within metabolic and signalling pathways. It is conceivable that this coordination might be assured by what we term functioning-dependent structures (FDSs), namely those assemblies of proteins that associate with one another when performing tasks and that disassociate when no longer performing them. To investigate a role in coordination for FDSs, we have studied numerically the steady-state kinetics of a model system of two sequential monomeric enzymes, E(1) and E(2).

Introduction to the concept of functioning-dependent structures in living cells.

The assembly of proteins into larger structures may confer advantages such as increased resistance to hydrolytic enzymes. metabolite channelling, and reduction of the number of proteins or other active molecules required for cell functioning. We propose the term functioning-dependent structures (FDSs) for those associations of proteins that are created and maintained by their action in accomplishing a function, as reported in many experiments.