George
J. Zanazzi
2nd Year Graduate Student
Department:
Neurobiology & Behavior
Graduate Program: Neuroscience
Advisor: Dr. Gary Matthews
Abstract:
Title:
Mechanisms of Exocytosis at Tonic Synapses
Fast communication between neurons occurs at synapses, where
vesicles in the presynaptic terminal release transmitter onto the
target neuron in response to an influx of calcium. Signal propagation
is particularly rapid and sustained at the first two synapses in the
visual system; i.e., the output synapses of photoreceptors and bipolar
cells. The presynaptic terminals of these two neurons tonically release
transmitter at active zones containing ribbons, which are proteinaceous
organelles that tether vesicles. While the synaptic ribbon is essential
for visual function, its precise role in exocytosis is unclear. The
ribbon may act like a conveyor belt that moves vesicles to the active
zone. Alternatively, the ribbon may provide a scaffold for vesicles
to fuse with each other in response to depolarization. While compound
fusion is known to occur in several secretory cell types, definitive
proof for this mechanism in synaptic vesicle exocytosis is lacking.
To examine vesicle fusion mechanisms at retinal ribbon synapses, we
are generating transgenic zebrafish that express sypHy, a pH-sensitive
GFP attached to the lumenal end of synaptophysin, in cone photoreceptors
and bipolar cells under the control of a heat shock promoter. Additional
transgenic zebrafish are being generated that express sypHy under
the control of the mGluR6 promoter, which we have found to drive robust,
specific expression of EYFP in ON bipolar cells. In order to visualize
sypHy-tagged vesicles specifically at synaptic ribbons, we are utilizing
a fluorescent peptide that binds to RIBEYE, a specific component of
ribbons. If homotypic vesicle fusion occurs at these synapses in response
to depolarization, there should be an increase in fluorescence along
the ribbon.
Membrane
fusion is driven by a core SNARE complex that appears to be clamped,
by a protein called complexin, in a fusion-ready state before calcium
enters the presynaptic terminal and binds to synaptotagmin. Complexins
3 and 4 are enriched at ribbon-containing synapses in the mammalian
retina, but their functions at these synapses are unknown. We have
identified two zebrafish complexin 3 orthologs and two complexin 4
orthologs in the retina. All four orthologs are expressed in the retina
by 5 days post-fertilization, coincident with the onset of visual
responses in zebrafish. In particular, complexins were found to be
robustly expressed in the outer and inner nuclear layers. To understand
the roles of these complexins in regulating vesicle fusion, we will
knockdown their expression with morpholino antisense oligos in transgenic
zebrafish that express sypHy. Since complexins found at conventional
synapses are thought to inhibit fast, synchronous exocytosis until
they are displaced by synaptotagmin, it is possible that ribbon-associated
complexins stabilize primed vesicles and clamp homotypic and heterotypic
fusion before stimulation. Taken together, these studies aim to characterize
cellular and molecular mechanisms of vesicle fusion at tonic synapses
in the retina, and may shed light on general mechanisms of exocytosis.
Publications:
(MSTP-supported publications indicated with an *)
*Zanazzi G, Matthews G. (2007). A doubleheader in
endocytosis. Neuron 56:939-942.
Koticha, D., Maurel, P., Zanazzi, G., Kane-Goldsmith,
N., Basak, S., Babiarz, J., Salzer, J., Grumet, M. (2006). Neurofascin
interactions play a critical role in clustering sodium channels, ankyrin
G and beta IV spectrin at peripheral nodes of Ranvier. Devel.
Biol. 293:1-12.
Melendez-Vasquez, C., Carey, D.J., Zanazzi, G., Reizes,
O., Maurel P., and Salzer, J.L. (2005). Differential expression of
proteoglycans at central and peripheral nodes of Ranvier. Glia.
52:301-308.
Taveggia C., Zanazzi, G., Petrylak, A., Yano, H.,
Rosenbluth, J., Einheber, S., Xu, X., Esper, R., Loeb, J., Shrager,
P., Falls, D.L., Chao, M.V., Role, L., and Salzer, J.L. (2005). Type
III neuregulin-1 levels determine the ensheathment fate of axons.
Neuron. 47:681-694.
Gil,
O.D., Zhang, L., Chen, S., Ren, Y.Q., Pimenta, A., Zanazzi,
G., Hillman, D., Levitt, P. and Salzer, J.L. (2002). Complementary
expression and heterophilic interactions between IgLON family members
neurotrimin and LAMP. J. Neurobiol. 51:190-204.
Rambukkana,
A., Zanazzi, G., Tapinos, N. and Salzer, J.L. (2002).
Contact-dependent induction of demyelination by Mycobacterium leprae
in the absence of immune cells. Science.
296:927- 931.
Chen,
S., Gil, O.D., Ren, Y.Q., Zanazzi, G., Salzer, J.L.
and Hillman, D. (2002). Neurotrimin expression during cerebellar development
suggests roles in axon fasciculation and synaptogenesis. J.
Neurocytol. 30:927-937.
Lustig
M.*, Zanazzi G.*, Sakurai T., Blanco C., Levinson
S.R., Lambert S., Grumet M., and Salzer J.L. (2001). Nr-CAM and neurofascin
interactions regulate ankyrin G and sodium channel clustering at the
node of Ranvier. Curr. Biol. 11:1864-1869.
*Equal contribution.
Melendez-Vasquez,
C., Rios, J.C., Zanazzi, G., Bretscher, A., Lambert,
S. and Salzer, J.L. (2001). Nodes of Ranvier form in association with
ERM-positive Schwann cell microvilli. Proc. Natl. Acad.
Sci. 98:1235-1240.
Zanazzi,
G., Einheber, S., Westreich, R., Hannocks, M.-J., Bedell-Hogan,
D., Marchionni, M.A. and Salzer, J.L. (2001). Glial growth factor/neuregulin
inhibits myelination and induces demyelination. J. Cell
Biol. 152:1289-1299.
Ng, V.*,
Zanazzi, G.*, Timpl, R., Talts, I.F., Salzer, J.L.,
Brennan, P.J. and Rambukkana, A. (2000). Role of the cell wall phenolic
glycolipid-1 in the peripheral nerve predilection of Mycobacterium
leprae. Cell. 103:511-524.
*Equal contribution.
Tikoo,
R., Zanazzi, G., Shiffman, D., Salzer, J. and Chao,
M.V. (2000). Cell cycle control of Schwann cell proliferation: role
of cyclin-dependent kinase-2. J. Neurosci.
20:4627-4634.
Ching, W.,
Zanazzi, G., Levinson, S.R. and Salzer, J.L. (1999).
Clustering of neuronal sodium channels requires contact with myelinating
Schwann cells. J. Neurocytol. 28:295-301.
Galbiati,
F., Volonte, D., Gil, O., Zanazzi, G., Salzer, J.L.,
Sargiacomo, M., Scherer, P.E., Engelman, J.A., Schlegel, A., Parenti,
M., Okamoto, T. and Lisanti, M.P. (1998). Expression of caveolin-1
and -2 in differentiating PC12 cells and dorsal root ganglion neurons:
caveolin-2 is up-regulated in response to cell injury. Proc.
Natl. Acad. Sci. 95:10257-10262.
Rambukkana,
A., Yamada, H., Zanazzi, G., Mathus, T., Salzer,
J.L., Yurchenco, P.D., Campbell, K.P. and Fischetti, V.A. (1998).
Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium
leprae. Science. 282:2076-2079.
Gil,
O.D., Zanazzi, G., Struyk, A. and Salzer, J.L. (1998).
Neurotrimin mediates bifunctional effects on neurite outgrowth via
homophilic and heterophilic interactions. J. Neurosci.
18:9312- 9325.
Einheber,
S., Zanazzi, G., Ching, W., Scherer, S., Milner,
T.A., Peles, E. and Salzer, J.L. (1997). The axonal membrane protein
Caspr/neurexin IV is a component of the septate-like paranodal junctions
that assemble during myelination. J. Cell Biol.
139:1495-1506.
Desser,
T.S., Rubin, D.L., Muller, H.H., Qing, F., Khodor, S., Zanazzi,
G., Young, S.W., Ladd, D.L., Wellons, J.A., Kellar, K.E.,
Toner, J.L. and Snow, R.A. (1994). Dynamics of tumor imaging with
Gd-DTPA-polyethylene glycol polymers: dependence on molecular weight.
J. Mag. Res. Imag.
4:467-472.
Young, S.W., Sidhu, M.K., Qing, F., Muller, H.H., Neuder, M., Zanazzi,
G., Mody, T.D., Hemmi, G., Dow, W., Mutch, J.D., Sessler,
J.L. and Miller R.A. (1994). Preclinical evaluation of gadolinium
(III) texaphyrin complex: a new paramagnetic contrast agent for magnetic
resonance imaging. Invest. Radiol. 29:330-338.
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