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.
"Many in vitro data have been published using N-terminal , , ,  or C-terminal , , , , ,  fluorescent tagged MMR proteins. However, fluorescent labeling might have significant influence on the functionality of tagged proteins , , . "
[Show abstract][Hide abstract] ABSTRACT: The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
PLoS ONE 02/2012; 7(2):e31863. DOI:10.1371/journal.pone.0031863 · 3.23 Impact Factor
"For Western blot, HEK293 cells were lysed in 20 mM HEPES, 1 mM EDTA, 0.1 g/L PMSF, 100 mM NaCl, 1% NP40, pH 7.4, with protease inhibitor cocktail (Calbiochem, San Diego, USA) , . Proteins were solubilized in SDS-PAGE buffer. "
[Show abstract][Hide abstract] ABSTRACT: Neurotransmitter release is elicited by an elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). The action potential triggers Ca(2+) influx through Ca(2+) channels which causes local changes of [Ca(2+)](i) for vesicle release. However, any direct role of extracellular Ca(2+) (besides Ca(2+) influx) on Ca(2+)-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.
Using photolysis of caged Ca(2+) and caffeine-induced release of stored Ca(2+), we found that extracellular Ca(2+) inhibited exocytosis following moderate [Ca(2+)](i) rises (2-3 µM). The IC(50) for extracellular Ca(2+) inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca(2+) concentration ([Ca(2+)](o)) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca(2+)](o). The calcimimetics Mg(2+), Cd(2+), G418, and neomycin all inhibited exocytosis. The extracellular Ca(2+)-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.
As an extension of the classic Ca(2+) hypothesis of synaptic release, physiological levels of extracellular Ca(2+) play dual roles in evoked exocytosis by providing a source of Ca(2+) influx, and by directly regulating quantal size and release probability in neuronal cells.
PLoS ONE 10/2011; 6(10):e24573. DOI:10.1371/journal.pone.0024573 · 3.23 Impact Factor
"For example, actin fused to GFP cannot function as the sole actin source in yeast (Doyle and Botstein, 1996). GFP fused to the carboxyl terminus of synaptotagmin cannot rescue a synaptotagmin deletion, whereas expression of wild-type synaptotagmin can rescue (Han et al., 2005). GFP fusions to either the amino or carboxyl terminus of BCRP, a drug resistance protein, are not functional (unpublished observation). "
[Show abstract][Hide abstract] ABSTRACT: The ability to localize proteins of interest in live cells through imaging inherently fluorescent protein tags has provided an unprecedented level of information on cellular organization. However, there are numerous cases where fluorescent tags alter the localization and/or function of the proteins to which they are appended. Clathrin-mediated endocytosis from the plasma membrane is a physiologically important process evolutionarily conserved from yeast to humans. Some proteins that are associated with the machinery of clathrin-mediated endocytosis have been tagged with fluorescent proteins. However, it has not yet been possible to study this process through a protein marker that is specific to this step and still fully functional when linked to a fluorescent protein. In this study, we present the first demonstration that one of these proteins, in this case a green fluorescent protein (GFP) fusion to alpha-adaptin, a marker of the adaptor protein-2 complex, functionally complements knockdown of endogenous protein through small interfering RNA silencing. GFP-alpha-adaptin, as well as the techniques used to test the fusion protein, represents an important contribution to the cell biologist's toolbox, which will permit a greater understanding of vesicle trafficking in live cells.
Amandine Dufour, Astrid Rollenhagen, Kurt Sätzler, Joachim H R Lübke,
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