Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca(2+) triggers exocytosis. The existence of sub-micrometer domains of greater than 100 microM [Ca(2+)](i) was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca(2+) 'microdomains' in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca(2+) nano- and microdomains forming around the mouth of voltage-gated Ca(2+) channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca(2+) channels at different central synapses and pointed out the role of Ca(2+) syntillas - localized, store operated Ca(2+) signals - in facilitation and spontaneous release. The coupling between Ca(2+) increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca(2+) comes from different sources, including Ca(2+) influx via the plasma membrane and the mobilization of Ca(2+) from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca(2+) signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca(2+) signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca(2+) sources, leading to a wide spectrum of Ca(2+) signals triggering release.     

Calcium sensors in regulated exocytosis

Neurotransmitter release, hormone secretion and a variety of other secretory process are tightly regulated with exocytotic fusion of secretory vesicles being triggered by a rise in cytosolic Ca2+ concentration. A series of proteins that act as part of a conserved core machinery for vesicle docking and fusion throughout the cell have been identified. In regulated exocytosis this core machinery must be controlled by Ca(2+)-sensor proteins that allow rapid activation of the fusion process following elevation of cytosolic Ca2+ concentration. The properties of such Ca2+ sensors are known from physiological studies but their molecular identity remains to be unequivocally established. The multiple Ca(2+)-dependent steps in the exocytotic pathway suggest the likely involvement of several Ca(2+)-binding proteins with distinct properties. Functional evidence for the role of various Ca(2+)-binding proteins and their possible sites of action is accumulating but a definitive identification of the major Ca(2+)-sensor in the final step of Ca(2+)-triggered membrane fusion in different cell types awaits further analysis.     

Calcium microdomains in aspiny dendrites

Dendritic spines receive excitatory synapses and serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity. Dendrites of GABAergic interneurons have few or no spines and thus do not possess a clear morphological basis for synapse-specific compartmentalization. We demonstrate using two-photon calcium imaging that activation of single synapses on aspiny dendrites of neocortical fast spiking (FS) interneurons creates highly localized calcium microdomains, often restricted to less than 1 microm of dendritic space. We confirm using ultrastructural reconstruction of imaged dendrites the absence of any morphological basis for this compartmentalization and show that it is dependent on the fast kinetics of calcium-permeable (CP) AMPA receptors and fast local extrusion via the Na+/Ca2+ exchanger. Because aspiny dendrites throughout the CNS express CP-AMPA receptors, we propose that CP-AMPA receptors mediate a spine-free mechanism of input-specific calcium compartmentalization.     

Late events in regulated exocytosis

To understand the intracellular mechanisms that control exocytosis it is necessary to have access to the cell interior. This is achieved by plasma membrane permeabilisation or by application of patch-pipettes. These conditions permit control over the cytosol composition and also allow leakage of soluble factors that may have roles in the exocytotic mechanism. Different permeabilisation methods allow different extents of leakage and therefore provide complementary data. The exocytotic machinery itself remains intact and can be activated by providing Ca2+ and/or a guanine nucleotide. In some cells there is evidence for the participation of two guanine nucleotide-binding proteins (GP and GE), as well as a Ca(2+)-binding protein. In others Ca2+ is the only requirement. In a number of cell types, ATP is not required for the late steps in the secretory pathway.     

Calcium microdomains in mitochondria and nucleus

Endomembranes modify the progression of the cytosolic Ca(2+) wave and contribute to generate Ca(2+) microdomains, both in the cytosol and inside the own organella. The concentration of Ca(2+) in the cytosol ([Ca(2+)](C)), the mitochondria ([Ca(2+)](M)) and the nucleus ([Ca(2+)](N)) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca(2+) from the high [Ca(2+)](C) microdomains generated during cell activation near Ca(2+) channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca(2+) signal to the bulk cytosol. This shaping of [Ca(2+)](C) signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca(2+)](M) signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca(2+)](C) signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca(2+)](N) signals could be generated by direct release of stored Ca(2+) into the nucleoplasm. Ca(2+) release could even be restricted to subnuclear domains. Putative Ca(2+) stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca(2+) into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain.     

Calcium microdomains and oxidative stress

The phenomenon of calcium microdomains is firmly established in the field of subcellular physiology. These regions of localized, transient calcium increase are exemplified by the spontaneous 'sparks' released through the ryanodine receptor in myocytes, but include subplasmalemmal microdomains, focal calcium oscillations and microdomains enclosed within organelles, such as the endoplasmic reticulum, golgi and mitochondria. Increasing evidence suggests that oxidative stress regulates both the formation and disappearance of microdomains. Calcium release channels and transporters are all modulated by redox state, while several mechanisms that generate oxidative or nitrosative stress are regulated by calcium. Here, we discuss the evidence for the regulation of calcium microdomains by redox state, and, by way of example, demonstrate that the frequency of calcium sparks in cardiomyocytes is increased in response to oxidative stress. We consider the evidence for the existence of analogous microdomains of reactive oxygen and nitrogen species and suggest that the refinement of imaging techniques for these species might lead to similar concepts. The interaction between Ca(2+) microdomains and proteins that modulate their formation results in a complex and dynamic, spatial signaling mechanism, which is likely to be broadly applicable to different cell types, adding new dimensions to the calcium signaling 'toolkit'.     

Calcium microdomains and cell cycle control

The cell division cycle comprises successive rounds of genome replication and segregation that are never error-free. A complex signalling network chaperones cell cycle events to ensure that cell cycle progression does not occur until any errors detected are put right. The signalling network consists of cell cycle control proteins that are phosphorylated and dephosphorylated, synthesized and degraded interactively to generate a set of sensors and molecular switches that are thrown at appropriate times to permit or trigger cell cycle progression. In early embryos, discrete calcium signals have been shown to be a key component of the molecular switch mechanism. In somatic cells in contrast, the participation of calcium signals in cell cycle control is far from clear. Recent experiments in syncytial Drosophila embryos have shown that localised calcium signals in the nucleus and mitotic spindle can be detected. It appears that the nucleus comprises a calcium signalling microdomain bounded by endoplasmic reticulum that isolates the nucleus and spindle. These findings offer a possible explanation for the apparent absence of calcium signals in somatic cells during mitosis.     

Calcium signaling complexes in microdomains of polarized secretory cells

The highly polarized nature of epithelial cells in exocrine glands necessitates targeting, assembly into complexes and confinement of the molecules comprising the Ca(2+) signaling apparatus, to cellular microdomains. Such high degree of polarized localization has been shown for all Ca(2+) signaling molecules tested, including G protein coupled receptors and their associated proteins, Ca(2+) pumps, Ca(2+) influx channels at the plasma membrane and Ca(2+) release channels in the endoplasmic reticulum. Although the physiological significance of polarized Ca(2+) signaling is clear, little is known about the mechanism of targeting, assembly and retention of Ca(2+) signaling complexes in cellular microdomains. The present review attempts to summarize the evidence in favor of polarized expression of Ca(2+) signaling proteins at the apical pole of secretory cells with emphasis on the role of scaffolding proteins in the assembly and function of the Ca(2+) signaling complexes. The consequence of polarized enrichment of Ca(2+) signaling complexes at the apical pole is generation of an apical to basal pole gradient of cell responsiveness that, at low physiological agonist concentrations, limits Ca(2+) spikes to the apical pole, and when a Ca(2+) wave occurs, it always propagates from the apical to the basal pole. Our understanding of Ca(2+) signaling in microdomains is likely to increase rapidly with the application of techniques to controllably and selectively disrupt components of the complexes and apply high resolution recording techniques, such as TIRF microscopy to this problem.     

Calcium regulation of exocytosis in PC12 cells

The calcium (Ca(2+)) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca(2+) was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca(2+) was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca(2+)-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca(2+) sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca(2+)-binding were not able to enhance release.     

调控型分泌的分子机制研究进展

调控型分泌途径对于维持内分泌细胞和神经元的功能非常重要。内分泌和神经系统的细胞将神经递质、神经肽和激素等包装在分泌囊泡内,然后在受到刺激时将这些物质释放到细胞外。调控型分泌囊泡,从生成、转运到与细胞膜的融合都需要许多蛋白质的参与和调节。简要总结参与这些过程的一些重要蛋白质的研究进展。     

Getting a G--RRP on regulated exocytosis in the heart

A study by Rybkin et al. (see p. 527) substantially advances our understanding of regulated exocytois by specialized secretory cells, such as atrial myocytes. A second member of the Ras-related protein family, RRP17, was identified and shown to participate in regulating the secretion of the cardiac-derived peptide hormone, atrial natriuretic peptide. In addition to the heart, RRP17 was shown to be expressed in neuronal, pancreatic, and skeletal muscle cells, suggesting a widespread role in regulated secretion for this new protein.     

Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains

Neuronal cell death is an early pathological feature of diabetic retinopathy. We showed previously that insulin receptor signaling is diminished in retinas of animal models of diabetes and that downstream Akt signaling is involved in insulin-mediated retinal neuronal survival. Therefore, further understanding of the mechanisms by which retinal insulin receptor signaling is regulated could have therapeutic implications for neuronal cell death in diabetes. Here, we investigate the role of cholesterol-enriched membrane microdomains to regulate PKC-mediated inhibition of Akt-dependent insulin signaling in R28 retinal neurons. We demonstrate that PKC activation with either a phorbol ester or exogenous application of diacylglycerides impairs insulin-induced Akt activation, whereas PKC inhibition augments insulin-induced Akt activation. To investigate the mechanism by which PKC impairs insulin-stimulated Akt activity, we assessed various upstream mediators of Akt signaling. PKC activation did not alter the tyrosine phosphorylation of the insulin receptor or IRS-2. Additionally, PKC activation did not impair phosphatidylinositol 3-kinase activity, phosphoinositide-dependent kinase phosphorylation, lipid phosphatase (PTEN), or protein phosphatase 2A activities. Thus, we next investigated a biophysical mechanism by which insulin signaling could be disrupted and found that disruption of lipid microdomains via cholesterol depletion blocks insulin-induced Akt activation and reduces insulin receptor tyrosine phosphorylation. We also demonstrated that insulin localizes phosphorylated Akt to lipid microdomains and that PMA reduces phosphorylated Akt. In addition, PMA localizes and recruits PKC isotypes to these cholesterol-enriched microdomains. Taken together, these results demonstrate that both insulin-stimulated Akt signaling and PKC-induced inhibition of Akt signaling depend on cholesterol-enriched membrane microdomains, thus suggesting a putative biophysical mechanism underlying insulin resistance in diabetic retinopathy.     

Calcium gradients and exocytosis in bovine adrenal chromaffin cells

The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small synchronous increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.     

A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.     

Acrosomal exocytosis, a special type of regulated secretion

The acrosome is a single secretory granule present in the head of mammalian--and other animal groups--sperm. Secretion of this granule is an absolute requirement for physiological fertilization. Acrosome exocytosis is a synchronized and tightly regulated all-or-nothing process, with no recycling of membranes. In the last few years, it has been shown that acrosomal exocytosis is mediated by a molecular mechanism that is homologous to that reported in the secretion of neuroendocrinal cells. Moreover, because of its particular characteristics, acrosomal exocytosis is a unique mammalian model for the study of the different steps of the membrane fusion cascade. Combining results in intact and permeabilized sperm, the following sequence of events has been proposed. In resting sperm, SNARE proteins are locked in inactive cis complexes. Sperm activation causes a calcium increase in the cytoplasm that promotes the production of cAMP and activates Rab3A. Afterwards, NSF and alphaSNAP disassemble cis complexes and the free SNAREs are then able to reassemble in loose trans complexes. Membrane fusion is arrested at this stage until calcium is released from inside the acrosome by inositol 1,4,5-trisphosphate-sensitive calcium channels to trigger the final steps of membrane fusion, which require fully assembled trans SNARE complexes and the calcium sensor synaptotagmin. This working model is still incomplete and tentative. Its improvement will be important to share light on this and other processes of regulated exocytosis. Moreover, it will bring new perspectives into the field of sperm-related fertility and sterility.