Calcium-independent exo-endocytosis coupling at small central synapses

At presynaptic terminals, neurotransmitters are released by synaptic vesicle exocytosis at the active zone. In order to maintain efficient neurotransmission and proper synaptic structure, sites of vesicle fusion must be cleared rapidly by endocytosis. Therefore, the coupling of exo- and endocytosis is crucial. Despite many years of research, the exact molecular and biophysical requirements for the coupling of exo- and endocytosis remain unclear. We investigate whether endocytosis can be triggered in a calcium-independent fashion by evoking calcium-independent exocytosis using a hypertonic sucrose solution. We demonstrate that endocytosis can be triggered, in the absence of calcium influx, in a clathrin-independent manner that relies on actin polymerization. Our findings point to a central role of membrane tension dependent on actin for efficient coupling of exo- and endocytosis

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr2+ triggers neurotransmitter release similar to Ca2+, but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca2+- and Sr2+-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C2B domain of synaptotagmin 1 bind Ca2+ in phospholipid complexes, but only the C2B domain forms Sr2+/phospholipid complexes; therefore, Sr2+ binding to the C2B domain is sufficient to trigger fast release, although with decreased efficacy. Ca2+ induces binding of the synaptotagmin C, domains to SNARE proteins, whereas Sr2+ even at high concentrations does not. Thus, triggering of the fast component of release by Sr2+ as a Ca2+ agonist involves the formation of synaptotagmin/ phospholipid complexes, but does not require stimulated SNARE binding

Critical role for Piccolo in synaptic vesicle retrieval

Loss of function of the presynaptic active zone protein Piccolo has recently been linked to a devastating disease causing brain atrophy. Here, we address how Piccolo inactivation adversely affects synaptic function and thus may contributes to neuronal loss. Our analysis shows that Piccolo is critical for the activity dependent recycling and maintenance of synaptic vesicles (SVs). Specifically, we find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by the reduced synaptic recruitment of Pra1, known to interact selectively with the zinc fingers of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to both developmental and psychiatric disorders

Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all gamma-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Muncl 3 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13- 2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity