Neuronal signaling occurs through both action potential-triggered synaptic vesicle fusion and spontaneous release, although the fusion clamp machinery that prevents premature exocytosis of synaptic vesicles in the absence of calcium is unknown. Here we demonstrate that spontaneous release at synapses is regulated by complexin, a SNARE complex-binding protein. Analysis of Drosophila melanogaster complexin null mutants showed a marked increase in spontaneous fusion and a profound overgrowth of synapses, suggesting that complexin functions as the fusion clamp in vivo and may modulate structural remodeling of neuronal connections by controlling the rate of spontaneous release.

The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550). Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike ⌬ctrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division. Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and DivK, and that phosphoryl transfer is not DivL's essential cellular activity.

The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Analysis of the requirements for CtrA polar localization and CtrA proteolysis revealed that both processes require a motif within amino acids 1-56 of the CtrA receiver domain, and neither process requires CtrA phosphorylation. These results strongly suggest that CtrA polar localization is coupled to its cell cycle-regulated proteolysis. The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Mutations in the divJ and pleC histidine kinases perturb the characteristic asymmetry of CtrA localization and proteolysis in the predivisional cell. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address.Caulobacter ͉ CtrA ͉ ClpXP I n Caulobacter crescentus, DNA replication occurs once per cell division, and the phases of the cell cycle are linked to observable morphological changes (reviewed in refs. 1 and 2). Motile swarmer cells (Fig. 1) in the G 1 phase of the cell cycle develop into stalked cells ( Fig. 1) and initiate chromosome replication. During the swarmer-to-stalked cell (SW-ST) (or G 1 -S) transition, each cell sheds its polar flagellum and builds a stalk at the same site. As DNA replication proceeds, stalked cells elongate and become predivisional cells with distinct poles. A new flagellum is built at the pole opposite the stalk, so that each cell division produces a motile swarmer cell and a stalked cell. Before cell separation, a diffusion barrier is established between the swarmer and stalked compartments of the predivisional cell so that they contain distinct sets of signal transduction proteins that yield progeny with different replicative fates (3): the stalked progeny can initiate DNA replication immediately, whereas the swarmer cell must first differentiate into a new stalked cell.A key signal transduction protein that regulates Caulobacter cell cycle progression is the essential response regulator CtrA (4). CtrA directly induces or represses the transcription of Ϸ55 operons in the Caulobacter genome, including genes needed for flagellum and pili biosynthesis, DNA methylation, cell division, chemotaxis, and metabolism (5, 6). However, CtrA also represses chromosome replication by binding to five sites within the replication origin (7). Because CtrA has multiple functions, its activity is tightly controlled by three mechanisms: cell cycleregulated transcription (...

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Synaptic transmission insures neuronal communication but relies on several random steps. Because synapse are still inaccessible to direct experimental recordings, to study synaptic reliability, we analyze synaptic transmission by constructing a biophysical model. This model accounts for the synaptic cleft geometry and several dynamical variables such as the position of vesicular release and the membrane trafficking AMPA receptors, which mostly mediate the synaptic current. These receptors are located in the postsynaptic terminal, but can be exchanged from the Post-Synaptic Density (PSD), a fundamental microdomain and the extra-synaptic space. We show that the synapse geometry controls the amplitude of the synaptic current, while receptor diffusional motion can replace desensitized receptors and thus prevents synaptic depression from receptor desensitization (significantly only after 6 to 7 successful spikes). Synaptic reliability is optimal when the active zone of vesicular release is apposed to a PSD, where AMPA receptors are concentrated. Change in this co-localization can lead to drastic effects on the synaptic current, which suggests that these changes can underlie a form of remodeling and plasticity. We finally demonstrate that fast temporal correlated spike lead to a reduced synaptic current. We conclude that although synapse should and are unreliable devices, at the neuronal level, reliability is restored due to the presence of multiple synaptic boutons.