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. Here we use two different systems, crystals of glycine and mixtures of proteins, to show that the formation of recombinant CN secondary ions under Cs bombardment can be exploited to create a new colocalization technique. We show experimentally that the formation of the recombinant (13)C(15)N secondary ion between (13)C- and (15)N-labeled macromolecules is indeed an indicator of the distance between the interacting macromolecules and on their shape. We build up a convolution model of the mixing-recombination process in D-SIMS that allows quantitative interpretations of the distance-dependent formation of the recombinant CN. Our results show that macromolecules can be colocalized if they are within 2 nm of one another. We discuss the potential advantages of this new technique for biological applications.

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.

BackgroundThere 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 hypothesisIn the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels.Testing the hypothesisEvidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding.Implications of the hypothesisThe physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined.