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.
|Clavijo Jordan, M Veronica; Lo, Su-Tang; Chen, Shiuhwei et al. (2016) Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate. Proc Natl Acad Sci U S A 113:E5464-71|
|Mitchell, Ryan K; Hu, Ming; Chabosseau, Pauline L et al. (2016) Molecular Genetic Regulation of Slc30a8/ZnT8 Reveals a Positive Association With Glucose Tolerance. Mol Endocrinol 30:77-91|
|Li, Daliang; Liu, Lin; Li, Wen-Hong (2015) Genetic targeting of a small fluorescent zinc indicator to cell surface for monitoring zinc secretion. ACS Chem Biol 10:1054-63|
|Li, Daliang; Huang, ZhiJiang; Chen, Shiuhwei et al. (2015) GLP-1 Receptor Mediated Targeting of a Fluorescent Zn(2+) Sensor to Beta Cell Surface for Imaging Insulin/Zn(2+) Release. Bioconjug Chem 26:1443-50|
|Hodson, David J; Mitchell, Ryan K; Marselli, Lorella et al. (2014) ADCY5 couples glucose to insulin secretion in human islets. Diabetes 63:3009-21|
|Hodson, David J; Mitchell, Ryan K; Bellomo, Elisa A et al. (2013) Lipotoxicity disrupts incretin-regulated human Î² cell connectivity. J Clin Invest 123:4182-94|
|Wu, Jiahui; Liu, Lin; Matsuda, Tomoki et al. (2013) Improved orange and red CaÂ²Â± indicators and photophysical considerations for optogenetic applications. ACS Chem Neurosci 4:963-72|
|Li, Wen-hong; Li, Daliang (2013) Fluorescent probes for monitoring regulated secretion. Curr Opin Chem Biol 17:672-81|
|Li, Wen-hong; Zheng, Genhua (2012) Photoactivatable fluorophores and techniques for biological imaging applications. Photochem Photobiol Sci 11:460-71|
|Schumacher, Jennifer A; Hsieh, Yi-Wen; Chen, Shiuhwei et al. (2012) Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans. Development 139:4191-201|
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