The ability of neurons to communicate with each other is fundamental for neural function. Information transfer occurs via neurotransmitters that are released from presynaptic terminals by Ca2+-evoked synaptic vesicle exocytosis and act on receptors in postsynaptic membranes. In addition to triggering exocytosis, Ca2+ regulates the efficiency of release in processes of presynaptic plasticity that mediate some forms of information processing in the brain. Our laboratory is interested in elucidating the mechanisms of neurotransmitter release and its regulation through analysis of the three-dimensional structure and interactions of proteins involved in this process. This research is not only relevant to neurotransmission but also to protein sorting and secretion in general, since most types of intracellular membrane traffic share a common membrane fusion machinery.
Our studies use a wide range of biophysical techniques that include multidimensional NMR spectroscopy, X-ray crystallography, cryo-electron microscopy and fluorescence spectroscopy among others. Our philosophy is to use whichever technique is most suitable to address specific, critical questions that arise as we and others keep making progress in shedding light onto the mechanisms of neurotransmitter release and intracellular membrane fusion in general. This work is performed in close collaboration with several laboratories, in particular those of Thomas Sudhof and Christian Rosenmund, where a variety of neurobiological approaches, including mouse genetics and electrophysiology, are being applied to study neurotransmitter release. The combined expertise from our laboratories allows us to study the mechanism of release using a wide range of techniques, in an endeavor where the structural studies are driven by specific biological questions and the results of these studies in turn drive the biology.
The primary targets of our research can be divided in two groups: i) components of the universal membrane fusion machinery such as the Sec1/Munc18 (SM) homologue munc18-1, the small Rab GTPase Rab3, and the SNARE proteins syntaxin 1, SNAP-25 and synaptobrevin; and ii) proteins with specialized roles in release such as synaptotagmin 1, complexin, munc13 and RIM. Our research has provided key insights into the structures, interactions and functions of these proteins, but we are still far from understanding the mechanisms underlying the different steps that lead to neurotransmitter release, i.e. synaptic vesicle docking, priming of the fusion machinery to a release-ready state, and Ca2+-dependent membrane fusion. After having analyzed the structures and interactions of many of these proteins using soluble fragments that lacked transmembrane regions, we are now focusing on the reconstitution into membranes of the macromolecular assemblies that mediate the different steps of release. A key aspect of these current efforts is the development of methodologies to analyze in detail protein-protein interactions on lipid bilayers.
Selected publications
X. Shao, B. A. Davletov, R. B. Sutton, T. C. Sudhof and J. Rizo, ?A Bipartite Ca2+-Binding Motif in C2 Domains of Synaptotagmin and Protein Kinase C?, Science 273, 248-251 (1996).
X. Shao, C. Li, I. Fernandez, X. Zhang, T. C. Sudhof and J. Rizo, ?Synaptotagmin-Syntaxin Interaction: the C2-Domain as a Ca2+-Dependent Electrostatic Switch?, Neuron 18,133-142 (1997).
J. Ubach, X. Zhang, X. Shao, T. C. Sudhof and J. Rizo, ?Ca2+-binding to synaptotagmin: how many Ca2+ ions bind at the tip of a C2-domain??, EMBO J. 17, 3921-3930 (1998).
I. Fernandez, J. Ubach, X. Zhang, T. C. Sudhof and J. Rizo, ?Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A?, Cell 18, 841-849 (1998).
I. Dulubova, S. Sugita, S. Hill, M. Hosaka, I. Fernandez, T. C. Sudhof and J. Rizo, ?A conformational switch in syntaxin during exocytosis?, EMBO J. 18, 4372-4382 (1999).
R. Fernandez-Chacon, A. Konigstorfer, S. H. Gerber, J. Garcia, M. F. Matos, C. F. Stevens, N. Brose, J. Rizo, C. Rosenmund and T. C. Sudhof, ?Synaptotagmin I functions as a Ca2+-regulator of release probability?, Nature 410, 41-49 (2001).
I. Fernandez, D. Arac, J. Ubach, S. H. Gerber, O. Shin, Y. Gao, R. G. W. Anderson, T. C. Sudhof and J. Rizo, ?Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine?, Neuron 32, 1057-1069 (2001).
X. Chen, D. Tomchick, E. Kovrigin, D. Arac, M. Machius, T. C. Sudhof and J. Rizo, ?Three-dimensional structure of the complexin/SNARE complex?, Neuron 33, 397-409 (2002).
I. Dulubova, T. Yamaguchi, Y. Gao, S. W. Min, I. Huryeva, T. C. Sudhof and J.Rizo, ?How Tlg2p/syntaxin 16 ?snares? Vps45p?, EMBO J. 21, 3620-3631 (2002).
J. Garcia, S. H. Gerber, S. Sugita, T. C. Sudhof and J. Rizo, ?A conformational switch in the piccolo C2A-domain regulated by alternative splicing?, Nat. Struct. Molec. Biol. 11, 45-53 (2004).
I. Dulubova, X. Lou, J. Lu, I. Huryeva, A. Alam, R. Schneggenburger, T. C. Sudhof and J. Rizo, ?A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity??, EMBO J. 24, 2839-2850 (2005).
J. Basu, N. Shen, I. Dulubova, J. Lu, R. Guan, O. Guryev, N. V. Grishin, C. Rosenmund and J. Rizo, ?Identification of a minimal domain responsible for Munc13 activity?, Nat. Struct. Molec. Biol. 12, 1017-1018 (2005).
X. Chen, D. Arac, T.-M. Wang, C. J. Gilpin, J. Zimmerberg and J. Rizo, ?SNARE-mediated lipid mixing depends on the physical state of the vesicles?, Biophys. J. 90, 2062-2074 (2006).
D. Arac, X. Chen, H. A. Khant, J. Ubach, S. J. Ludtke, M. Kikkawa, A. E. Johnson, W. Chiu, T. C. Sudhof and J. Rizo, ?Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin 1 to phospholipids?, Nat. Struct. Molec. Biol. 13, 209-217 (2006).
Lu,J., Machius,M., Dulubova,I., Dai,H., Sudhof,T.C., Tomchick,D.R., and Rizo,J. Structural Basis for a Munc13-1 Homodimer to Munc13-1/RIM Heterodimer Switch. PLoS. Biol. 4, 1159-1172 (e192) (2006).
Rizo,J., Chen,X., and Arac,D. Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release. Trends Cell Biol. 16, 339-350 (2006).
Tang, J., Maximov, A., Shin, O.-H., Dai, H., Rizo, J. and Sudhof, T. C. A complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175-1187 (2006).
Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T. C. and Rizo, J. Munc18-1 binds directly to the neuronal SNARE complex. Proc. Natl. Acad. Sci. USA 104, 2697-2702 (2007).
Dai, H., Shen, N., Arac, D. and Rizo, J. A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release. J. Mol. Biol. 367, 848-863 (2007).
RESEARCH INTERESTS
Mechanisms of neurotransmitter release and presynaptic plasticity
Mechanisms of intracellular membrane fusion and its regulation
RECENT PUBLICATIONS
Dai, H., Shen, N., Arac, D. and Rizo, J., "A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release" J. Mol. Biol., 367:848-863, 2007
J. Basu, N. Shen, I. Dulubova, J. Lu, R. Guan, O. Guryev, N. V. Grishin, C. Rosenmund and J. Rizo, "Identification of a minimal domain responsible for Munc13 activity" Nat. Struct. Molec. Biol., 12:1017-1018, 2005
X. Chen, D. Arac, T.-M. Wang, C. J. Gilpin, J. Zimmerberg and J. Rizo, "SNARE-mediated lipid mixing depends on the physical state of the vesicles" Biophys. J., 90:2062-2074, 2006
Lu,J., Machius,M., Dulubova,I., Dai,H., Sudhof,T.C., Tomchick,D.R., and Rizo,J., "Structural Basis for a Munc13-1 Homodimer to Munc13-1/RIM Heterodimer Switch." PLoS. Biol., 4:1159-1172, 2006
Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T. C. and Rizo, J., "Munc18-1 binds directly to the neuronal SNARE complex" Proc. Natl. Acad. Sci. USA, 104:2697-2702, 2007
SIGNIFICANT PUBLICATIONS
X. Chen, D. Tomchick, E. Kovrigin, D. Arac, M. Machius, T. C. S|dhof and J. Rizo, "Three-dimensional structure of the complexin/SNARE complex" Neuron, 33:397-409, 2002
I. Fernandez, J. Ubach, X. Zhang, T. C. S|dhof and J. Rizo, "Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A" Cell, 18:841-849, 1998
X. Shao, B. A. Davletov, R. B. Sutton, T. C. S|dhof and J. Rizo, "Bipartite Ca2+-Binding Motif in C2 Domains of Synaptotagmin and Protein Kinase C" Science, 273:248-251, 1996
I. Dulubova, S. Sugita, S. Hill, M. Hosaka, I. Fernandez, T. C. S|dhof and J. Rizo, "A conformational switch in syntaxin during exocytosis" EMBO J., 18:4372-4382, 1999
D. Arac, X. Chen, H. A. Khant, J. Ubach, S. J. Ludtke, M. Kikkawa, A. E. Johnson, W. Chiu, T. C. Sudhof and J. Rizo, "Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin 1 to phospholipids" Nat. Struct. Molec. Biol., 13:209-217, 2006
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