The Photoconvertible Fluorescent Protein Dendra2 Tag as a Tool to Investigate Intracellular Protein Dynamics.

Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.

Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta.

BACKGROUND: Synaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid ß (Aß) peptide, and neurotoxic Aß42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aß production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer's disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins. RESULTS: Mass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca(2+)-dependent PS1 binding profiles in vitro and in vivo. We found that Aß level, and more critically, conformation of the PS1 and the Aß42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aß production at the synapse. Moreover, ß-secretase 1 (BACE1) stability, ß- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking. CONCLUSIONS: Our findings identify Syt1 as a novel Ca(2+)-sensitive PS1 modulator that could regulate synaptic Aß, opening avenues for novel and selective synapse targeting therapeutic strategies.

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells.

Neurons and certain kinds of endocrine cells, such as adrenal chromaffin cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles exhibit differences in Ca(2+) sensitivity and contain diverse signaling substances. The present work was undertaken to identify the synaptotagmin (Syt) isoforms present in secretory vesicles. Fractionation analysis of lysates of the bovine adrenal medulla and immunocytochemistry in rat chromaffin cells indicated that Syt 1 was localized in LDCVs and SLMVs, whereas Syt 7 was the predominant isoform present in LDCVs. In contrast to PC12 cells and the pancreatic ß cell line INS-1, Syt 9 was not immunodetected in LDCVs in rat chromaffin cells. Double-staining revealed that Syt 9-like immunoreactivity was nearly identical with fluorescent thapsigargin binding, suggesting the presence of Syt 9 in the endoplasmic reticulum (ER).The exogenous expression of Syt 1-GFP in INS-1 cells, which had a negligible level of endogenous Syt 1, resulted in an increase in the amount of Syt 9 in the ER, suggesting that Syt 9 competes with Syt 1 for trafficking from the ER to the Golgi complex. We conclude that LDCVs mainly contain Syt 7, whereas SLMVs contain Syt 1, but not Syt 7, in rat and bovine chromaffin cells.

Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals.

Memory and learning in animals are mediated by neurotransmitters that are released from vesicles clustered at the synapse. As a synapse is used more frequently, its neurotransmission efficiency increases, partly because of increased vesicle clustering in the presynaptic neuron. Vesicle clustering has been believed to result primarily from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system, and the postsynaptic cell. Our in vivo experiments on the embryonic Drosophila nervous system show that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension within the axons. Vesicle clustering vanishes upon severing the axon from the cell body, but is restored when mechanical tension is applied to the severed end of the axon. Clustering increases when intact axons are stretched mechanically by pulling the postsynaptic muscle. Using micro mechanical force sensors, we find that embryonic axons that have formed neuromuscular junctions maintain a rest tension of approximately 1 nanonewton. If the rest tension is perturbed mechanically, axons restore the rest tension either by relaxing or by contracting over a period of approximately 15 min. Our results suggest that neuromuscular synapses employ mechanical tension as a signal to modulate vesicle accumulation and synaptic plasticity.

Lipid rafts association of synaptotagmin I on synaptic vesicles.

We confirmed the raft association of synaptotagmin I (syt I) in synaptic vesicles by sucrose density gradient centrifugation, cholesterol depletion, and temperature dependence, and Ca2+ was found to positively regulate this association. Furthermore, using syt I mutants we found that the transmembrane domain (TMD) of syt I plays an important role in localizing syt I into the lipid rafts of synaptic vesicles, and the raft association of the TMD can be regulated by its phosphorylation status.

Dvl regulates endo- and exocytotic processes through binding to synaptotagmin.

Dvl, an important component of the Wnt signalling pathway, is thought to be involved in synaptogenesis. In this study, we investigated whether Dvl regulates neurotransmitter release. Knockdown of Dvl in PC12 cells suppressed K(+)-induced dopamine release, and this phenotype was restored by expression of Dvl-1. We identified synaptotagmin (Syt) I, which is involved in neurotransmitter release, as a Dvl-binding protein. Dvl directly bound to the C2B domain of Syt I. Dvl colocalized with Syt I at the tip of neurites of differentiated PC12 cells and of neurons in the rat dorsal root ganglion. Dvl and Syt I was located in large dense-core vesicles, which contain dopamine. In addition, endocytosis of vesicles containing Syt I was suppressed in Dvl knockdown PC12 cells. Dvl inhibited the binding of Syt I to the complex consisting of syntaxin-1A and SNAP-25. Furthermore, micro2-adaptin of AP-2, which is known to play a role in endocytosis, formed a complex with Dvl and Syt I. Taken together, these results suggest that Dvl is involved in endo- and exocytotic processes through the binding to Syt I.