The overall goal of these studies is to elucidate the membrane trafficking pathway that is used in the recycling of synaptic vesicles (SVs) in living synapses. According to previous data, SV recycling could either involve two sequential budding events, a primary budding from the plasma membrane and a secondary budding from endosomes, or only a single budding step from the plasma membrane. It has not been possible to distinguish between these two possibilities in previous studies, primarily due to difficulties to determine whether internal """"""""endosome-like"""""""" structures seen in ultrathin sections represent endosomes or plasma membrane invaginations. These difficulties can be overcome by using high voltage electron microscopic (HVEM) examination of thicker sections combined with electron tomography to determine precisely 3D relationships between structures. We will apply HVEM to examine the 3D ultrastructure of presynaptic membrane structures in specimen blocks (1-2 5m semithin sections) that include the entire synaptic region of the giant reticulospinal synapse in lamprey. This synapse has been extensively characterized and proved suitable as a model to study vesicle recycling mechanisms. By applying high frequency action potential stimulation, the synaptic vesicle pool can be depleted. The recovery of the vesicles will be examined in specimens fixed at different time points following cessation of stimulation. To visualize putative endosomal intermediates that may be difficult to observe under physiological conditions, we will use protocols that perturb synaptic vesicle endocytosis at different steps. These include microinjection of proteins and nucleotides that interfere with the fission of coated pits, and perfusion of calcium-free solution which arrests the onset of the formation of clathrin coated pits. The application of HVEM and tomographic reconstruction to the lamprey model will make it possible to detail the microanatomy of synaptic vesicle recycling and thereby to elucidate a membrane trafficking pathway that is of key importance in synaptic transmission. Work on this project is underway.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Diego
La Jolla
United States
Zip Code
Funakoshi, Shunsuke; Miki, Kenji; Takaki, Tadashi et al. (2016) Enhanced engraftment, proliferation, and therapeutic potential in heart using optimized human iPSC-derived cardiomyocytes. Sci Rep 6:19111
Rubio-Marrero, Eva N; Vincelli, Gabriele; Jeffries, Cy M et al. (2016) Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1. J Biol Chem 291:5788-802
Yin, Xinghua; Kidd, Grahame J; Ohno, Nobuhiko et al. (2016) Proteolipid protein-deficient myelin promotes axonal mitochondrial dysfunction via altered metabolic coupling. J Cell Biol 215:531-542
Zhao, Claire Y; Greenstein, Joseph L; Winslow, Raimond L (2016) Roles of phosphodiesterases in the regulation of the cardiac cyclic nucleotide cross-talk signaling network. J Mol Cell Cardiol 91:215-27
Rajagopal, Vijay; Bass, Gregory; Walker, Cameron G et al. (2015) Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes. PLoS Comput Biol 11:e1004417
Schachtrup, Christian; Ryu, Jae Kyu; Mammadzada, Könül et al. (2015) Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-? signaling and astrocyte functions. Nat Neurosci 18:1077-80
Sanders, Matthew A; Madoux, Franck; Mladenovic, Ljiljana et al. (2015) Endogenous and Synthetic ABHD5 Ligands Regulate ABHD5-Perilipin Interactions and Lipolysis in Fat and Muscle. Cell Metab 22:851-60
Takeshima, Hiroshi; Hoshijima, Masahiko; Song, Long-Sheng (2015) Ca²? microdomains organized by junctophilins. Cell Calcium 58:349-56
Mills, Elizabeth A; Davis, Chung-ha O; Bushong, Eric A et al. (2015) Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis. Proc Natl Acad Sci U S A 112:10509-14
Kim, K-Y; Perkins, G A; Shim, M S et al. (2015) DRP1 inhibition rescues retinal ganglion cells and their axons by preserving mitochondrial integrity in a mouse model of glaucoma. Cell Death Dis 6:e1839

Showing the most recent 10 out of 384 publications