Although a typical cell measures only tens of micrometers in diameter, it is a highly organized and spatially heterogeneous structure. Cell membrane receptors and proteins receive extracellular stimulations and transform them into intracellular signals, signals that dictate the behavior of the cell and errors in the propagation of these signals underlie a wide range of diseases. The distribution of receptors and proteins on the cell membrane determines and affects the rate and pattern of the propagation of these intracellular signals. Membrane proteins are often expressed in different morphological regions of the plasma membrane, and within a local area, the proteins tend to form clusters and patches, rather than distribute homogeneously and continuously on the membrane. To study and dissect the mechanism and signaling pathways by which a single neuronal cell processes the arrival of a particular signal at its membrane surface, we aim to develop a technique by which a precisely timed stimulus or set of stimuli can be delivered to the cell with high spatial (sub-micrometers) and temporal (sub- microseconds) resolution. Such a tool will find use for functional mapping of cell surface proteins and for probing the dynamics of signal transduction and synaptic transmission triggered by the localized activation of receptors. This tool is based both on the ability to synthesize nanoscale capsules and to release the confined molecules from select capsules with a single laser pulse. To develop and demonstrate this method, we have the following aims: (1) Photosensitize the shell of nanocapsules with near-IR dyes or with chromophores that have high two-photon absorption cross sections, so we may use near-IR or two-photon laser pulses to trigger release, (2) Develop new nanocapsules having homogeneous sizes, and which are tunable from ~20nm to ~100nm; these nanocapsules also should be able to encapsulate a wide range of molecules (from small neurotransmitters to peptides and proteins) at high concentrations and should exhibit long-term storability, and (3) Map neuronal response caused by release of physically caged amyloid-? 42 (A?-42). A?-42 is believed to be the causative factor in Alzheimer disease, although its mechanism of action is poorly understood; here we propose to study the spatial-temporal dynamics of neuronal activation by A?-42. Cells respond to their environment through a complex and interdependent series of signal transduction pathways that frequently begin at the cell membrane with high spatial and temporal resolutions (e.g. exocytosis, endocytosis, synaptic transmission). To study and dissect the mechanism and signaling pathways by which a cell processes the arrival of a particular signal at its membrane surface, we propose here to develop a method by which a precisely timed stimulus can be delivered to the cell with high spatiotemporal resolution. This method will facilitate the detailed study of signaling pathways that underlie disease processes, such as Alzheimer that is a devastating neurological disease associated with aging. ? ? ?
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