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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1.

To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1- enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.[1]

References

  1. C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1. Han, W., Rhee, J.S., Maximov, A., Lin, W., Hammer, R.E., Rosenmund, C., Südhof, T.C. J. Biol. Chem. (2005) [Pubmed]
 
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