Calmodulin, synchronous and asynchronous release of neurotransmitter

Evidence collected from studies on a wide range of secretory cells suggests that calmodulin may play an important role in stimulus-secretion coupling. Work on synaptosomes, central synaptic preparations and chromaffin cell preparations indicates that calmodulin probably also acts as the intracellular Ca2+-receptor for secretion in neuronal cells, Ca2+-binding resulting in activation of protein kinases and phosphorylation of certain secretory vesicle proteins. Studies on the effects of calmodulin-binding drugs at peripheral synapses have given surprising results, particularly the finding that evoked (synchronous) transmitter release is not suppressed by calmodulin inhibition, though asynchronous release can be markedly inhibited. It is suggested that the insensitivity of synchronous release to drug treatment is due to the fact that only vesicle-bound calmodulin is involved in this form of transmitter secretion. Asynchronous release, however, involves recruitment of cytosolic calmodulin and can therefore be inhibited by calmodulin-binding drugs.     

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.     

Synaptotagmin在神经递质释放过程中的作用

神经突触间递质的释放是神经系统完成其生理功能最重要的生物现象之一。在贮存递质的突触囊泡上存在一些神经细胞所特有的囊泡蛋白,如突触素（synapsin）、synaptobrevin和synaptotagmin等。其中synaptotagmins是膜转运蛋白中的一个家族,它们的特征是含有两个钙结合区：C2A和C2B。到目前为止,在哺乳动物中已经发现了15种synaptotagmin同形物（isoforms）。神经递质释放是由Ca^2＋内流以诱导突触囊泡发生胞吐而引起的,Ca^2＋需与细胞内部的Ca^2＋感受器相结合来协同控制囊泡胞吐释放,SynaptotagminⅠ可能作为快速同步释放的Ca^2＋感受器而发挥作用。现在已知synaptotagminⅠ在胞吐和胞吞两个过程中都扮演重要角色。     

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

Expression and function of synaptotagmin VII in CTLs

The Ca(2+) sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca(2+)-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII(+/+) controls, Syt VII(-/-) animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII(-/-) mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII(+/+) or Syt VII(-/-) CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.     

Synthesis of photoreactivating enzyme in synchronous and asynchronous cultures of Escherichia coli

The technique of flash photolysis was used to study cellular variations in the number of photoreactivating enzyme (PRE) molecules during the cell division cycle of the UV-sensitive E. coli strain B_S?1. No variations in the number of PRE molecules per genome were observed throughout the cell division cycle when synchronized cells cultured in either glucose-minimal or succinate-minimal medium were used. This is interpreted to mean that PRE synthesis is continuous throughout the cell cycle for glucose-grown cells, but may stop at the time chromosome replication ceases prior to division, in succinate-grown cells. The effect of growth rate and stage of growth on cellular PRE content in asynchronous cultures was also determined. Variations in the number of PRE per genome were observed for both synchronous and asynchronous cells cultured in different media and occurred in a manner that suggested a dependence on growth rate. PRE per genome increased with generation time. Stationary phase cells from each culture medium (nutrient broth, glucose-minimal, succinate-minimal) had more PRE per genome than did respective log phase cells. It is suggested that PRE synthesis may be controlled by some aspect of chromosome replication.     

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.     

Study of asynchronous and synchronous yeast cultures using flow cytofluorometry]

Distribution of Saccharomyces cerevisiae, Candida boidinii and Candida tropicalis cells according to DNA content was investigated using laser flow cytofluorometry. Cells distribution curves according to DNA content possessed two maxima in case the sample belonged to the exponential phase of the asynchronous batch culture, or one maximum in case the sample was from the stationary phase of growth. In synchronous cultures variations of cells distribution curves according to DNA content (age structure of the population) were demonstrated and the curves with one maximum and plateau were observed.     

Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2, 3, 4, or 5 (day 0 = day of breeding) can be transferred with +/- 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2, 2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P less than .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P less than .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral environment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P less than .01) and young born (56% vs. 19%, P less than .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

Calcium regulation of spontaneous and asynchronous neurotransmitter release

The molecular machinery underlying action potential-evoked, synchronous neurotransmitter release, has been intensely studied. It was presumed that two other forms of exocytosis, delayed (asynchronous) and spontaneous transmission, were mediated by the same voltage-activated Ca(2+) channels (VACCs), intracellular Ca(2+) sensors and vesicle pools. However, a recent explosion in the study of spontaneous and asynchronous release has shown these presumptions to be incorrect. Furthermore, the finding that different forms of synaptic transmission may mediate distinct physiological functions emphasizes the importance of identifying the mechanisms by which Ca(2+) regulates spontaneous and asynchronous release. In this article, we will briefly summarize new and published data on the role of Ca(2+) in regulating spontaneous and asynchronous release at a number of different synapses. We will discuss how an increase of extracellular [Ca(2+)] increases spontaneous and asynchronous release, show that VACCs are involved at only some synapses, and identify regulatory roles for other ion channels and G protein-coupled receptors. In particular, we will focus on two novel pathways that play important roles in the regulation of non-synchronous release at two exemplary synapses: one modulated by the Ca(2+)-sensing receptor and the other by transient receptor potential cation channel sub-family V member 1.     

Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine.

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts.

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

ASP-based method for the enumeration of attractors in non-deterministic synchronous and asynchronous multi-valued networks

Background

This paper addresses the problem of finding attractors in biological regulatory networks. We focus here on non-deterministic synchronous and asynchronous multi-valued networks, modeled using automata networks (AN). AN is a general and well-suited formalism to study complex interactions between different components (genes, proteins,...). An attractor is a minimal trap domain, that is, a part of the state-transition graph that cannot be escaped. Such structures are terminal components of the dynamics and take the form of steady states (singleton) or complex compositions of cycles (non-singleton). Studying the effect of a disease or a mutation on an organism requires finding the attractors in the model to understand the long-term behaviors.

Results

We present a computational logical method based on answer set programming (ASP) to identify all attractors. Performed without any network reduction, the method can be applied on any dynamical semantics. In this paper, we present the two most widespread non-deterministic semantics: the asynchronous and the synchronous updating modes. The logical approach goes through a complete enumeration of the states of the network in order to find the attractors without the necessity to construct the whole state-transition graph. We realize extensive computational experiments which show good performance and fit the expected theoretical results in the literature.

Conclusion

The originality of our approach lies on the exhaustive enumeration of all possible (sets of) states verifying the properties of an attractor thanks to the use of ASP. Our method is applied to non-deterministic semantics in two different schemes (asynchronous and synchronous). The merits of our methods are illustrated by applying them to biological examples of various sizes and comparing the results with some existing approaches. It turns out that our approach succeeds to exhaustively enumerate on a desktop computer, in a large model (100 components), all existing attractors up to a given size (20 states). This size is only limited by memory and computation time.