1987 — 1997 |
Fernandez, Julio M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Stimulus-Secretion Coupling in Mast Cells @ University of Pennsylvania
This project combines circuit analysis and patch-clamp techniques, to measure the cell membrane expansion (increase in surface area) associated with exocytosis, in single isolated rat peritoneal mast cells. This study will focus on a quantitative determination of the time course of membrane expansion after stimulation. Exocytosis will be monitored at two levels: i) Macroscopic measurements accurately follow the time course of the overall increase in the membrane area. This time course is represented by three parameters a lag period d, where no sizeable area increase is detected after stimulation, a time constant that represents the secretory granule fusion rate after exocytosis begins and the ratio of the final to initial area, A, that reflects the extent of degranulation. Various activators and inhibitors of exocytosis will be tested for their effect on these parameters and mechanistic models that account for the observed effects will be attempted. ii) Microscopic measurements follow unitary events of secretory granule fusion which are to be represented as a distribution of amplitude, and fusion rates. These data will help establish the nature of the various types of unitary events observed (exocytosis, endocytosis, other). Existing theories of exocytosis claim the involvement of various enzymes like phospholipase A2 (PLA2), polyphosphoinositide phosphodiesterase (PDE) or messengers like Ca++ ions, arachidonic acid, lysolipids or physical factors like osmotic pressure. Since the patch-clamp technique, as used in this project, allows for intracellular as well as extracellular perfusion of the cell under study, enzyme inhibitors and products can be directly applied to the cytosol and their effect quantified as in i) and ii). The same is true for the cell's physical environment where osmotic gradients and temperature are easily controlled. In summary, the project will attempt a quantitative description of exocytosis at the single cell level and use it to test for various theories of exocytosis. These results will help understanding the mechanisms of Antigen induced release of inflammatory mediators as well as the mechanism of exocytosis in general.
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0.911 |
1998 — 2002 |
Marszalek, Piotr Fernandez, Julio Oberhauser, Andres |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Force-Induced Conformational Transitions in Single Polysaccharide Molecules by Afm
MARSZALEK MCB9808310 Polysaccharide molecules have been found to behave as entropic springs with complex force-dependent elasticity. Polysaccharides can adopt a variety of secondary structures in solution due to different types of monomers and glycosidic linkages between the monomers (e.g. alpha-(1-4), beta (1-4), alpha-(1-6), etc.). This study examines the hypothesis that the elasticity of polysaccharides is related to their secondary structures, and seeks to identify force-induced conformational transitions that may abruptly affect the length and elasticity of these molecules. Towards these aims a representative group of linear polysaccharides that tend to adopt distinct secondary structures - extended and ribbon-like cellulosic chains, wide helical amylosic chains, and flexible dextran-like chains - will be investigated by stretching single molecules vertically in the atomic force microscope. Different derivatives of polysaccharides will be used to study how the steric and electrostatic effects of the substituted groups affects chain elasticity. The kinetics of the force-induced conformational transitions (continuous or discontinuous) will be probed by varying the rate at which extension of the molecule and the elastic force is generated. Molecular dynamics (MD) simulations of disaccharide segments will be carried out to investigate how an external mechanical force affects geometry of covalent bonds and conformations of the glucopyranose ring. This will help to identify the mechanism underlying enthalpic elasticity and possible force-induced conformational transitions. Information derived from the AFM experiments and MD calculations will be integrated to construct a kinetic model, using Monte Carlo simulation, of polysaccharide elasticity that will reproduce force-extension characteristics. The elastic and viscoelastic properties of polysaccharides are widely exploited in nature and they have many industrial applications. The proposed studies will generate valuable inform ation on the atomic basis of polysaccharide elasticity. In addition, deciphering the nature of conformational transitions in stretched polysaccharides may shed light on the mechanisms of force-induced transitions in more complex macromolecules, such as DNA.
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0.91 |
2013 — 2016 |
Fernandez, Julio |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Idbr: High Throughput Single Molecule Afm Force-Spectrometer
An award has been made to Columbia University to develop the first high throughput single molecule AFM force-spectrometer. Single-molecule force spectroscopy by atomic force microscopy (AFM) is widely used to measure the dynamic of proteins placed under force, of common occurrence in Biology. A growing number of biologists are taking advantage of the detailed information that emerges from force-spectroscopy measurements. However, current force-spectrometers face huge limitations since they are remarkably low-throughput. The aims of the proposal are designed to overcome the limitations of AFM spectrometers and aims to construct a new instrument where throughput is increased by at least two orders of magnitude. Together with the construction of the new instrument, novel technologies for the covalent attachment of the molecules will be developed which will allow for long-term recordings (hours to days) from a single protein molecule placed under force. In order to analyze the large volume of data anticipated, new methods of analysis based on Extended Kalman Filters will be implemented. By increasing at least by a factor of 100 the throughput of force-spectroscopy by AFM, new capabilities will be accessible to a wide community of Biologists interested in protein dynamics, protein mechanics and other disciplines. Currently, there is a growing consensus that mechanical perturbations of proteins are commonplace in vivo. However, it is extremely challenging even to conceive experiments to find small molecules that affect the mechanical properties of a protein, such as mechanical stability. Such compounds would be extraordinarily useful to understand the role of mechanical forces in physiology and disease, and they may even find therapeutic application. Being able to test hundreds of small molecules in a working day will allow the identification of such small molecules.
This interdisciplinary proposal will be used to train undergraduate, graduate and postdoctoral students in the art of instrument design, and single molecule recordings. Both undergraduate and graduate students will be actively involved in the design and construction of the high-throughput force spectrometer. In particular, undergraduate students always find interdisciplinary research very attractive and make use of summer research programs to engage in interdisciplinary projects, such as the one described in this proposal. A graduate student and a postdoctoral fellow will work full time in different aspects of the proposal. The proposal includes a mentoring plan for the postdoctoral fellow that will ease his transition to an independent position, increasing his chances to become a successful member of the research community. The dissemination of the results is crucial to the success of the proposal. The ultimate goal of the proposal is to make the new instrumentation available through partnerships with companies, in combination with presentations in conferences and workshops.
Funded through the Instrument Development for Biological Research program in the Division of Biological Infrastructure.
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