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GENERATION OF TRANSGENIC MICE FOR IN VIVO DETECTION OF INSULIN-CONTAINING GRANULE exocytosis.AND QUANTIFICATION OF INSULIN SECRETION

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Journal of Innovative Optical Health Sciences 2009年第4期2卷 397-405页

作者： JINLING LU NATALIA GUSTAVSSON QIMING LI GEORGE KRADDA THOMAS C.SUUDHOF WEIPING HAN Laboratory of Metabolic Medicine Singapore Bioimaging Consortium A*STAR Singapore 138667 Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute Stanford University School of Medicine Palo Alto CA 94304USA Department of Biochemistry Yong Loo Lin School of Medicine National University of Singapore Singapore 117597

Insulin secretion is a complex and highly regulated *** much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living *** few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in *** combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in *** this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the *** exocytosis.of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory ***,mCherry fluorescence enabled tracking the movement of secretory granules in living *** validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-*** transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.

关键词： Insulin secretion diabetes optical imaging secretory granule exocytosis.

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Microtubule Depolymerization Affects Endocytosis and exocytosis.in the Tip and Influences Endosome Movement in Tobacco Pollen Tubes

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Molecular Plant 2013年第4期6卷 1109-1130页

作者： Aurora Irene Idilli Piero Morandini Elisabetta Onelli Simona Rodighiero Marco Caccianiga Alessandra Moscatelli Department of Biosciences Milan University Via Celoria 26 20133 Milan Italy Fondazione Filarete Milan University Viale Ortles 22 Milan Italy

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments （AFs） and microtubules （MTs）. AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane （PM）. Secretory activity is balanced by PM retrieval that maintains cell mem- brane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching （FRAP） experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis.in the central domain of the apical PM.

关键词： pollen tube microtubules endocytosis exocytosis.

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Modes of Exocytotic and Endocytotic Events in Tobacco BY-2 Protoplasts

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Molecular Plant 2011年第2期4卷 241-251页

作者： Vera Bandmann Marko Kreft Ulrike Homann Institut fur Botanik Technische Universitat Darmstadt 64287 Darmstadt Germany Laboratory of Neuroendocrinology and Molecular Cell Physiology University of Ljubljana and Celica Biomedical Center Technology Park 24 1000 Ljubljana Slovenia

To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cellattached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events, fusion occurred transiently, which facilitates rapid recycling of vesicles. In addition, transient sequential or multi-vesicular exocytosis.observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together, the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.

关键词： Electrophysiology cell walls protein traffic and secretion BY-2 endocytosis exocytosis.

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