The long-term goal of the proposed research is to understand the mechanisms by which Ca2+ regulates mitotic progression in cells. To achieve this goal, novel fluorescent probes of Ca2+ and Ca2+-dependent signalling processes will be synthesized and applied in mitotic cells. Two types of probes are proposed for synthesis: 1) a non-metabolizable fluorescent aqueous-soluble polymer-supported Ca2+ indicator which based on fura-2. A polymer-bound indicator, by evading cellular transport mechanisms, can be used for monitoring Ca2+ concentration in mitotic cells over the entire course of mitosis without artifacts arising from extrusion or sequestration of the indicator into subcellular organelles. 2) Fluorescent peptide probes that specifically detect the activation of protein kinase C, multifunctional Ca2+/calmodulin-dependent protein kinase, and calmodulin. Each peptide is a specific recognition sequence, either for phosphorylation by a specific kinase or for binding to calmodulin, flanked by independently synthesized fluorescent amino acid residues that form a donor-acceptor pair for resonant energy transfer (RET). RET efficiency will be sensitive to the peptide conformation which , in turn, changes in response to phosphorylation or calmodulin binding. By measuring RET in a specific peptide probe spectroscopically, one can monitor activation of calmodulin or a specific kinase. Application of these probes in mitotic cells will yield information on the pathways by which Ca2+ exerts influence on various stages of mitosis.
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