Molecular Mechanisms of Ca2+-triggered Exocytosis
Our research is focused on understanding the structure, function and dynamics of the exocytotic membrane "fusion machine" that mediates the release of neurotransmitters from neurons. These studies have begun to reveal insights into how the release machinery is regulated and thereby contributes to neuronal plasticity.
Neuronal exocytosis is triggered by Ca2+ and occurs via the abrupt opening of a pre-assembled fusion pore. Subsequent dilation of the pore results in the complete fusion of the vesicle membrane with the plasma membrane. We are currently identifying and reconstituting the sequential protein-protein and protein-lipid interactions that underlie excitation-secretion coupling. To delineate this pathway, we have primarily focused on the Ca2+-binding synaptic-vesicle protein, synaptotagmin, which appears to function as the Ca2+-sensor that regulates release. Our work is also focused on components of the "SNARE-complex", which is thought to form the core of the fusion apparatus, and on a number of additional regulatory proteins. The rapid kinetics of exocytosis (<1 ms) indicate that only a handful of molecular rearrangements occur to couple Ca2+-synaptotagmin to the opening of the fusion pore. We are using a number of cell biology, genetic and biophysical approaches (FRET, TIRF, AFM, FM dyes, patch clamp, amperometry, voltammetry etc.) to delineate the interactions/conformational changes that occur during this window of time. Current experiments include the modulation of fusion pores in cultured neurons and model cell lines, reconstitution of Ca2+-triggered membrane fusion in vitro, and the visualization of protein rearrangements in vitro and inside living cells. We are in the process of expanding these studies to understand how changes in fusion pores affects local synaptic circuitry. Another main focus of the laboratory concerns the mechanism by which clostridial neurotoxins - the agents that cause botulism and tetanus poisoning - enter neurons to block exocytosis.
- Yao, J, Gaffaney, J.D., Kwon, S.E. and Chapman, E.R.. (2011). Doc2 is a Ca2+-sensor required for asynchronous neurotransmitter release. Cell 147(3):666-77. PMID: 22036572 [PMCID - in process]
- Yao, J., Kwon, S.E., Gaffaney, J.D., Dunning, F.M., and Chapman, E.R.. (2011). Uncoupling the roles of synaptotagmin I as a dual Ca2+ sensor during endocytosis and exocytosis of synaptic vesicles. Nature Neuroscience 5(2): 243-9. PMID: 22197832 [PMCID - in process]
- Kwon, S.E. and Chapman, E.R.. (2011). Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons. Neuron 70(5): 847-54. PMCID: PMC3136197.
- Hui, E., Gaffaney, J.C., Wang, Z., Johnson, C., Evans, C. and Chapman, E.R. (2011). Mechanism and function of synaptotagmin-mediated membrane apposition. Nature Struct. Mol. Biol. 18(7): 813-821. PMCID: PMC3130839
- Sun, S., Suresh, S., Liu, H., Tepp, W.H., Johnson, E.A., Edwardson, J.M., Chapman, E.R.. (2011). Probing the assembly of a botulinum neurotoxin translocation channel. Cell Host & Microbe 10(3):237-47. PMID: 21925111 [PMCID - in process]
- Wang, Z., Liu, H., Gu, Y. and Chapman, E.R... (2011). Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion. J. Cell Biol. 195(7):1159-70. PMID: 22184197 [PMCID - in process]
- Yeh, F., Zhu, Y., Tepp, W.H., Johnson, E.A., Bertics, P.J., Chapman, E.R.. (2011). Retargeting clostridial neurotoxins to macrophages reduces TNFα release. Biochemistry 50(48):10419-21. PMCID PMC3226321.
- Wu, Y., Ma, L., Cheley, S., Bayley, H., Cui, Q. and Chapman, E.R.. (2011). Permeation of styryl dyes through nanometer-scale pores in membranes. Biochemistry 50(35): 7493-502. PMID: 21815625 [PMCID - in process]
- Wang, D., Zhang, Z., Dong, M., Sun, S., Chapman, E.R., and Jackson, M.B. (2011). Syntaxin requirement for Ca2+-triggered exocytosis in neurons and endocrine cells demonstrated with an engineered neurotoxin. Biochemistry 50(14): 2711-3. PMID: 21401123 [PMCID - in process]
- Paddock, B.E., Wang, Z., Biela, L.M., Chen, K., Getzy, M.D., Striegel, A., Richmond, J.E., Chapman, E.R., Featherstone, D.E. and Reist, N.E. (2011). Membrane penetration by synaptotagmin is required for coupling calcium binding to vesicle fusion in vivo. J. Neurosci. 31(6): 2248-57. PMCID: PMC3092483
- Mayers, J.R., Olendrowitz, C., Schuh, A.L., Chapman, E.R., Eimer, S. and Audhya, A.. Multiple roles for Hrs and STAM in multivesicular endosome dynamics. (2011). J. Biol. Chem. 86(11):9636-45. PMID: 21193406 [PMCID - in process]
- Zhang, Z., Wu, Y., Wang, Z., Dunning, F.M., Rehfuss, J., Ramanan, D., Chapman, E.R., and Jackson, M.B.. (2011). Release mode of large and small dense-core vesicles specified by different synaptotagmin isoforms in PC12 cells. Mol. Biol. Cell 22(13): 2324-36. PMCID: PMC3128534