Edwin R. Chapman

 

 

Molecular Mechanisms of Ca2+-triggered Exocytosis

E-mail: chapman@physiology.wisc.edu

Research Strengths: Membrane Excitability and Synaptic Transmission, Molecular Neuroscience

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.

Website:

http://www.physiology.wisc.edu/www/chapman.html

Lab Website:

http://www.physiology.wisc.edu/chapman/

Selected Publications:

• *Chai, Q., *J.W. Arndt, *M. Dong, W.H. Tepp, E.A. Johnson, E.R. Chapman and R.C. Stevens. 2006. Structural basis of receptor recognition by botulinum neurotoxin B. Nature In press.
  *equal contribution
• Dong, M., Y. Yeh, W.H. Tepp, C. Dean, E.A. Johnson, R. Janz, and E.R. Chapman. 2006. SV2 is the protein receptor for botulinum neurotoxin A. Science (published on line 10.1126/science.1123654)
• *Bhalla, A., *M.C. Chicka, W.C. Tucker, and E.R. Chapman. 2006. Ca2+*synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. In press.
  *equal contribution
• Jackson, M.B. and E.R. Chapman. 2006. Fusion pores and fusion machines in Ca2+-triggered exocytosis. Ann. Rev. Biophys. Biomol. Struct. In press.
• Wang, C.T., J. Bai, P.Y. Chang, E.R. Chapman, and M.B. Jackson. 2006. Synaptotagmin*Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation. J. Physiol. 570: 295-307.
• Liu, T., W. Tucker, A. Bhalla, E.R. Chapman, and J.C. Weisshaar. 2005. SNARE-driven, 25-millisecond vesicle fusion in vitro. Biophysical J. 89: 2458-2472.
• Wang, P., M.C. Chicka, A. Bhalla, D. Richards, and E.R. Chapman. 2005. Synaptotagmin VII is targeted to secretory organelles in PC12 cells where it functions as a high affinity calcium sensor. Mol. Cell. Biol. 25: 8693-8702.
• Bhalla, A., W. Tucker, and E.R. Chapman. 2005. Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+ and Sr2+ concentration to SNARE-mediated membrane fusion. Mol. Biol. Cell 16: 4755-4764.
• Hui, E., J. Bai, M. Sugimori, R. Llinas, and E.R. Chapman. 2005. Three distinct kinetic groupings of the synaptotagmin family - candidate sensors for rapid and delayed exocytosis. Proc. Natl. Acad. Sci. U.S.A. In press.
• Richards, D., J. Bai, and E.R. Chapman. 2005. Two modes of exocytosis revealed by the rate of FM1-43 efflux in hippocampal boutons. J. Cell Biol. 168: 929-939.
• Shen, S.S., W.C. Tucker, E.R. Chapman, and R.A. Steinhardt. 2005. Molecular regulation of membrane resealing in 3T3 fibroblasts. J. Biol. Chem. 280: 1652-60.
• Maher, B., R.L. MacKinnon II, J. Bai, E.R. Chapman, and P.T. Kelly. 2005. Activation of intracellular Ca2+ stores modulates glutamate receptor cycling in hippocampal neurons. J. Neurophysiol. 93: 178-188.
• Dong, M., M.C. Goodnough, W.H. Tepp, E.A. Johnson, and E.R. Chapman. 2004. Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells. Proc. Natl. Acad. Sci. U.S.A. 101: 14701-14706. [PDF]
• Grishanin, R.N., J.A. Kowalchyk, V.A. Klenchin, K-S. Ann, C.A. Earles, E.R. Chapman, R.R.L. Gerona, and T.F.J. Martin. 2004. CAPS acts at a pre-fusion step in dense-core vesicle exocytosis as a PIP2-binding protein. Neuron 43: 551-562. [PDF]
• Tucker, W.T., T. Weber, and E. R. Chapman. 2004. Reconstitution of Ca2+-triggered membrane fusion by synaptotagmin and SNAREs. Science 304: 435-438. [PDF]
• Han, X., C.T. Wang, J. Bai, E.R. Chapman, and M.B. Jackson. 2004. Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Science 304: 289-292. [PDF]
• Bai, J., C.T. Wang, , M.B. Jackson, and E.R. Chapman. 2004. Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions. Neuron 41: 929-942. [PDF]
• Bai, J., W.C. Tucker, and E.R. Chapman. 2004. PIP2 increases the speed-of-response of synaptotagmin and steers its membrane penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11: 36-44. [PDF]
• Bai, J. and E.R. Chapman. 2004.The C2-domains of synaptotagmin: Partners in exocytosis. TIBS 29: 143-151.
• Chieregatti, E., M.C. Chicka, E.R. Chapman, and G. Baldini. 2004. SNAP-23 functions in docking/fusion of granules at low Ca2+. Mol. Biol. Cell. 15: 1918-30.
• Berdiev, B.K., B. Jovov, H.L.R. Ganeshan, W. Tucker, A.P. Naren, C.M. Fuller, E.R. Chapman, and D.J. Benos. 2004. The effects of syntaxin 1A on ENaC. AJP: Renal Physiology 286: F1100-F1106.
• Wang, C.T., J.C. Lu, J. Bai, T.F.J. Martin, E.R. Chapman, and M.B. Jackson. 2003. Different domains of synaptotagmin control the choice between kiss-and-run and full-fusion. Nature 424: 943-947. [PDF]
• Tucker, W.C., J.M. Edwardson, J. Bai, H.J. Kim, T.F.J. Martin, and E.R. Chapman. 2003. Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells. J. Cell Biol. 162: 199-209. [PDF]
• Edwardson, J.M., C.T. Wang, B. Gong, A. Wyttenbach, J. Bai, M.B. Jackson, E.R. Chapman, and A.J. Morton. 2003. Mutant huntingtin expression inhibits exocytosis in PC12 cells by depletion of complexin II. J. Biol. Chem. 278: 30849-30853.
• Bai, J. and E.R. Chapman. 2003. Application of fluorescent probes to determine the mechanics and dynamics of Ca2+-triggered synaptotagmin C2-domain-membrane interactions. Meth. Enzymol. 360: 238-258.
• Wu, Y., Y. He, B. Bai, S.R. Ji, W.C. Tucker, E.R. Chapman,* and S.F. Sui.* 2003. Visualization of synaptotagmin I oligomers assembled onto lipid monolayers. Proc. Natl. Acad. Sci. U.S.A. 100: 2082-2087. [PDF]
• Wang, P., C.T. Wang, J. Bai, M.B. Jackson, and E.R.Chapman. 2003. Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a non-additive manner. J. Biol. Chem. 278: 47030-47037. [PDF]
• Dong, M., M.C. Goodnough, W.H. Tepp, E.A. Johnson, and E.R. Chapman. 2003. Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells. J. Cell Biol. 162: 1293-1303.
• Liu, W., V. Montana, E.R. Chapman, U. Mohideen, and V. Parpura. 2003 . Botulinum Toxin type B micromechanosensor. Proc. Natl. Acad. Sci. USA 100: 13621-13625. [PDF]
  
  *corresponding authors.