The long term goal of this project is to engineer photonic probes to enable biological discovery. This proposal represents an integral approach by combining molecular design, organic synthesis, optogenetics, and advanced fluorescence microscopy to develop fluorescent probes and imaging techniques, and to apply them to study an important function of gap junction coupling: how gap junction intercellular communication mediates synchronized cell secretion. To this end, we will first develop a new class of fluorescent probes for imaging the dynamics of regulated exocytosis with very high sensitivity and spatiotemporal resolution. The development is based on the observation that a number of secretory cells, including pancreatic islet beta cells, contain a high level of zinc ion (Zn2+) in their secretory granules. Upon stimulation, these cells release the contents of their secretory granules into extracellular medium, during which Zn2+ is co-released. By engineering zinc sensors to specifically report local Zn2+ rise near plasma membranes, we are able to monitor Zn2+ granule release continuously at cellular and subcellular resolution. To examine how gap junction coupling regulates synchronized secretion, we will apply the technique of optogenetics to control the membrane excitability, and to integrate the method of photo-activation with zinc imaging. Combined with pharmacological and genetic approaches to manipulate cell coupling strength, we will investigate how gap junction coupling synchronizes cell secretion. Finally, to characterize how cells coordinate their secretory activity in physiological preparations or in tissues where normal cell-cell contact is maintained, we will use imaging methods of high spatial selectivity, including two photon laser scanning microscopy and spinning disk confocal microscopy, to examine Zn2+ granule release in three dimensions at cellular and subcellular resolution. New probes and methods developed here should have broad applications in cellular and neuronal biology and in different biological systems.
Abnormal cell coupling has been implicated in a number of diseases including cardiac arrhythmia, deafness, neuronal demyelination, and cataracts. Understanding mechanisms regulating cell junctional communication and its functions remains a significant biological challenge which has important implications in both health and disease. The focus of this proposal is to develop new imaging probes and techniques to study how gap junction coupling coordinates cell secretion.
|Hodson, David J; Mitchell, Ryan K; Marselli, Lorella et al. (2014) ADCY5 couples glucose to insulin secretion in human islets. Diabetes 63:3009-21|
|Li, Wen-hong; Li, Daliang (2013) Fluorescent probes for monitoring regulated secretion. Curr Opin Chem Biol 17:672-81|
|Hodson, David J; Mitchell, Ryan K; Bellomo, Elisa A et al. (2013) Lipotoxicity disrupts incretin-regulated human * cell connectivity. J Clin Invest 123:4182-94|
|Li, Wen-hong; Zheng, Genhua (2012) Photoactivatable fluorophores and techniques for biological imaging applications. Photochem Photobiol Sci 11:460-71|
|Li, Daliang; Chen, Shiuhwei; Bellomo, Elisa A et al. (2011) Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR). Proc Natl Acad Sci U S A 108:21063-8|
|Li, Wen-Hong (2010) Photo-activatable probes for the analysis of receptor function in living cells. Methods Mol Biol 591:105-20|
|Zheng, Genhua; Ambros, Victor; Li, Wen-Hong (2010) Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent. Silence 1:9|
|Ouyang, Xiaohu; Shestopalov, Ilya A; Sinha, Surajit et al. (2009) Versatile synthesis and rational design of caged morpholinos. J Am Chem Soc 131:13255-69|
|Wu, Zhuoru; Luby-Phelps, Katherine; Bugde, Abhijit et al. (2009) Capacity for stochastic self-renewal and differentiation in mammalian spermatogonial stem cells. J Cell Biol 187:513-24|
|Yang, Song; Li, Wen-Hong (2009) Assaying dynamic cell-cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP. Nat Protoc 4:94-101|
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