Cellular Regulators Part B: Calcium and Lipids

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However the functional importance of PS scrambling for secretion is still under debate and the precise kinetics of this translocation is not established. An interesting possibility lies in the fact that PS contributes substantially to the negative charge of the inner leaflet of the plasma membrane. Phosphoinositides are a class of phospholipids characterized by an inositol head group that can be phosphorylated on the three, four, and five positions to generate seven distinct species key in cell signaling and trafficking.

Much of the work carried out on exocytosis has focused on the role played by PtdIns 4,5 P 2. Indeed a number of pioneer studies indicated that PtdIns 4,5 P 2 positively modulates secretion in neuroendocrine cells 16 — Using patch clamp experiments on intact chromaffin cells and in parallel analyzing images of plasma membrane lawns, it was subsequently shown that over-expression of the kinase that generates PtdIns 4,5 P 2 causes an increase in the plasmalemmal PtdIns 4,5 P 2 level and secretion, whereas over-expression of a membrane-tagged PtdIns 4,5 P 2 phosphatase eliminates plasmalemmal PtdIns 4,5 P 2 and inhibits secretion Thus, the balance between the generation and degradation rates of the plasmalemmal PtdIns 4,5 P 2 directly regulates the extent of exocytosis from chromaffin cell.

Using the phosphatidylinositol 3-kinase inhibitor LY, a correlation between the level of the plasma membrane PtdIns 4,5 P 2 and the size of the primed vesicle pool was found 19 , Wen et al. Importantly, such an inhibition promotes a transient rise in PtdIns 4,5 P2 that was sufficient to mobilize secretory vesicles to the plasma membrane via activation of the small GTPase Cdc42 and actin polymerization.

More recently, a functional link between PtdIns 4,5 P 2 signaling and secretory vesicle dynamics through de novo remodeling of the actin cytoskeleton was also described These observations are consistent with a function of PtdIns 4,5 P 2 as an acute regulator of secretion. PtdIns 4,5 P 2 seems to lie in a key position controlling the size and refilling rate of the primed vesicle pools, but not the fusion rate constants per se.

In line with this model, we recently reported that the HIV PtdIns 4,5 P 2 -binding protein Tat is able to penetrate neuroendocrine cells and accumulate at the plasma membrane through its binding to PtdIns 4,5 P 2. By sequestering plasma membrane PtdIns 4,5 P 2 , Tat alters neurosecretion, reducing the number of exocytotic events without significantly affecting kinetic parameters fusion pore opening, dilatation, and closure of individual events Other phosphoinositides seem to act as signaling or recruitment factors to prime secretory vesicles for exocytosis.

For instance, experiments carried out on permeabilized chromaffin cells reveal that PtdIns 3 P located on a subpopulation of chromaffin granules positively regulates secretion 21 , Hence, these studies highlight a complex regulation of neuroexocytosis by phosphoinositides, with PtdIns 4,5 P 2 and PtdIns 3 P being essential factors promoting ATP-dependent priming in neurosecretory cells. It is intriguing that PtdIns 3,5 P 2 displays an opposite effect, but reveals how fine-tuning of exocytosis by phosphoinositides could potentially control the number of vesicles undergoing priming in response to a stimulation.

The local formation of PA is a recurring theme in intracellular membrane traffic and its involvement in regulated exocytosis has been suggested in various models, including neuroendocrine cells 14 , The development of molecular tools has enabled the identification of phospholipase D1 PLD1 as the key enzyme responsible for PA synthesis during exocytosis 14 , Capacitance recordings from chromaffin cells silenced for PLD1 suggest that PLD1 controls the number of fusion competent secretory granules at the plasma membrane without affecting earlier recruitment or docking steps, leading to the idea that PA acts directly in membrane fusion In agreement with this concept, a molecular sensor for PA revealed local PA accumulation at the plasma membrane near morphologically docked granules at sites of active exocytosis Various other lipids are suspected to take part in regulated exocytosis.

Although most of them have been implicated based on in vitro membrane fusion assays, some have also been studied in neuroendocrine cells. For instance, diacylglycerol DAG increases stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool through the regulation of the vesicle priming protein Munc Furthermore by activating protein kinase C, DAG may modulate the phosphorylation level of various proteins contributing or regulating the exocytotic machinery, including SNAP and Munc18 30 , Modulating PS levels also directly affects the rate of exocytosis in PC12 cells.

Finally, arachidonic acid produced from different phospholipids by phospholipase A2 and from DAG by DAG-lipase potentiates exocytosis from chromaffin cells 33 , Within membranes, the ability of microdomains to sequester specific proteins and exclude others makes them ideally suited to spatially organize cellular pathways. For instance, numerous studies of the distribution of SNARE proteins in various cell types suggest that SNAREs partially associate with detergent resistant, cholesterol-enriched microdomains Palmitoylation appears to be the major targeting signal in these microdomains, as in the case of SNAP, although it is likely that other elements contribute to the enrichment of constituents of the exocytotic machinery within these cholesterol microdomains.

However despite intense research there is still little known about what lipid or protein molecules are actually present at sites of exocytosis. Up to 20 proteins potentially involved in regulated exocytosis have been reported to bind PtdIns 4,5 P 2 Using immunogold labeling of plasma membrane sheets combined with spatial point pattern analysis, we recently observed that PtdIns 4,5 P 2 microdomains co-localize with SNARE clusters and docked secretory granules Translocation of the PtdIns 4,5 P 2 -binding protein annexin A2 to the plasma membrane following cell stimulation is a hallmark of chromaffin cell exocytosis Annexin A2 plays an essential role in calcium-regulated exocytosis by promoting PtdIns 4,5 P 2 and cholesterol-enriched domains containing SNAREs in the vicinity of docked granules 9 , Altogether these observation raise the notion that functional exocytotic sites defined by specific lipids such as cholesterol, GM1, and PtdIns 4,5 P 2 are able to recruit and sequester components to build a machine that drives fast and efficient membrane fusion Figure 1.


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Figure 1. Model highlighting the importance of lipids for membrane fusion. Molecular details of how PtdIns 4,5 P 2 forms a platform for vesicle recruitment have recently been proposed Like PtdIns 4,5 P 2 , these anionic lipids can probably recruit syntaxin-1A, and it is tempting to propose that the recruitment of syntaxin isoforms may depend on the type of lipid present. Furthermore, the fact that these lipids can be quickly converted into different forms using kinases, lipases, and phosphatases, such a protein-recruiting mechanism offers a supplementary level of control to adapt the exocytotic machinery to the physiological demands put on the cell.

Finally, using super-resolution optical techniques and fluorescence lifetime imaging microscopy, it was shown that distinct t-SNARE intermediate states on the plasma membrane can be patterned by the underlying lipid environment Undoubtedly these high-resolution imaging techniques will be useful to determine how lipids contribute to the organization of the exocytotic platform. PtdIns 4,5 P 2 directly binds to a large subset of proteins from the exocytotic machinery. Finally, PtdIns 4,5 P 2 controls actin polymerization by modulating the activity and targeting of actin regulatory proteins.

Indeed the activity of the actin-binding proteins scinderin and gelsolin, two F-actin severing proteins that are constituents of the exocytotic machinery, is regulated by PtdIns 4,5 P 2 A transient increase in PIP2 levels is sufficient to promote the mobilization and recruitment of secretory vesicles to the plasma membrane PIP2 therefore links exocytosis and the actin cytoskeleton by coordinating the actin-based delivery of secretory vesicles to the exocytotic sites.

Diacylglycerol production through hydrolysis of PtdIns 4,5 P 2 by phospholipase C is mandatory for exocytosis DAG is essential in the priming of exocytosis, owing to the activation of protein kinase C and Munc13, which then modulate the function of syntaxin-1A This pathway is essential for exocytosis as inhibition of DAG lipase blocks exocytosis PA directly activates these proteins, but evidence that this activation directly contributes to exocytosis remains scarce.

PA is also an essential cofactor of phosphatidylinositolphosphate 5-kinase, which produces PtdIns 4,5 P 2 , suggesting a possible positive feedback loop in the synthesis of PA and PtdIns 4,5 P 2 Although no direct evidence in neuroendocrine systems have shown that PA directly regulates the assembly or the function of the minimal fusion machinery, in vitro reconstituted fusion assays with purified yeast vacuolar SNAREs do so.

Interestingly, omega-3 and omega-6 fatty acids, which play important roles in human health, have be shown to recapitulate this in vitro effect of arachidonic acid on SNARE complex formation, suggesting that syntaxins may represent crucial targets of polyunsaturated lipids In other words, polyunsaturated lipids may physiologically regulate SNARE complex assembly and thus exocytosis.

Along this same line, sphingosine a releasable backbone of sphingolipids, activates vesicular synaptobrevin facilitating the assembly of SNARE complexes required for membrane fusion It is however important to note that the effects of arachidonic acid and sphingosine observed in these studies are all achieved near or at the CMC value for these lipids, treatments that may also lead to membrane disorganization like detergent action.

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The most widely accepted model for membrane fusion, the stalk pore model proposes that the merging of cis contacting monolayers gives rise to a negatively curved lipid structure called a stalk. The structure of this stalk depends on the composition of the cis monolayers the outer leaflet of the vesicle and the inner leaflet of the plasma membrane.

This model implies that cone-shaped lipids such as cholesterol, DAG, or PA, which have intrinsic negative curvatures, in the cis leaflets of contacting bilayers would enhance membrane fusion Vice versa inverted cone-shaped lipids such as PS, gangliosides, or lysophospholipids should prevent membrane fusion in the cis leaflets, but promote fusion when present in the outer leaflets Interestingly, GM1 was found enriched in the outer leaflet of the plasma membrane at the sites of exocytosis in stimulated chromaffin cells 9.

These GM1 domains may induce positive membrane curvature in the outer leaflet 55 , thereby promoting fusion Figure 1. Reconstituted fusion assays and direct addition of lipids on cultured cells validate the concept that PA, DAG, and cholesterol might promote membrane fusion by changing the spontaneous curvature of membranes [reviewed in 8 ]. At physiological concentrations, PtdIns 4,5 P 2 inhibits SNARE-dependent liposome fusion 45 , most likely due to its intrinsic positive curvature-promoting properties.

However, PtdIns 4,5 P 2 has been described to be converted from an inverted cone-shaped structure to a cone-shaped form in the presence of calcium Thus, in stimulated cells, a local accumulation of PA and PtdIns 4,5 P 2 at granule docking sites where GM1 is in the outer leaflets may well have a synergistic effect on membrane curvature and promote fusion Figure 1. In an alternate mode of changing membrane topology, synaptotagmin has been proposed to facilitate membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS It is worth to mention that most of lipid mentioned in this review, also have the ability to flip from one leaflet to another.

How this flipping is regulated and how it affects curvature remains an unsolved issue. However, it is likely that the ability of these lipids to interact with the fusion machinery largely controls these flipping properties. As illustrated in this review, a given lipid can play multiple functions, acting either individually or successively or even simultaneously in concert with other lipids. At the same time, the rapid enzymatic production and degradation of lipids at exocytotic sites allows the cell to remain flexible: by changing the lipid levels, physiological function can be modified within seconds or minutes without the need for protein synthesis or degradation.

Over the last decade, in vitro reconstituted membrane fusion combined with precise methods to quantify specific lipid species and improved molecular and pharmacological tools to manipulate cellular levels of a given lipid, have lead to a better understanding of the capacities of lipids to promote exocytosis at different steps of the process. For different kinds of vesicles in different cell types, it is likely that the local lipid environment may differentially regulate fusion pore formation, enlargement, and duration, which may in part explain the great variety of fusion kinetics observed in vivo.

Finally, lipids could also contribute to the tight coupling between exocytosis and the early stages of membrane retrieval and endocytosis as highlighted in a review of this issue Houy et al. Undoubtedly, the next challenge will be to follow individual lipid dynamics at the speed of pore formation and expansion. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We wish to thank Dr.

Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Nancy Grant for critical reading of the manuscript. Biochim Biophys Acta — Jahn R, Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles. Nature —7. Nature — SNAREpins: minimal machinery for membrane fusion. Cell 92 — CrossRef Full Text. Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release. Science —5. Martin TF.

Role of PI 4,5 P 2 in vesicle exocytosis and membrane fusion. Subcell Biochem 59 — Phospholipases and fatty acid signalling in exocytosis. J Physiol — Lipid dynamics in exocytosis. Cell Mol Neurobiol 30 — Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles.

Mol Biol Cell 16 — Lang T. J Physiol —8.

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Frontiers | Lipids in Regulated Exocytosis: What are They Doing? | Endocrinology

Traffic 11 — Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells. Traffic 12 — Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells. EMBO J 20 — Phospholipid scramblaseinduced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells. J Neurosci 33 — Aikawa Y, Martin TF. ARF6 regulates a plasma membrane pool of phosphatidylinositol 4,5 bisphosphate required for regulated exocytosis. Fig 1 — Diagram showing lipid metabolism.

The top right section of the diagram demonstrates the conversion of Acetyl-CoA to fatty acids. This occurs in the mitochondria and produces acetyl-CoA which can either enter the TCA cycle or be used to produce ketone bodies. The long chains of fatty acids are broken down into a series of 2 carbon acetate units, which are then combined with co-enzyme A to form acetyl-CoA.

This acetyl-CoA can then be combined with oxaloacetate to form citrate for the beginning of the TCA cycle. Glucagon and adrenaline stimulate the process of lipolysis whereas it is inhibited by insulin. Protein synthesis is stimulated by insulin and growth hormone.

Dynamics and functions of lipid droplets

The following are synthesised within the liver:. They are metabolised in the liver but the amino group is potentially toxic and must be removed. One option is transamination, where the amino group can be transferred to ketoacids through the actions of alanine aminotransferase ALT and aspartate aminotransferase AST :. The ammonia is then converted to an ammonium ion, which must be removed due to toxicity. It can be removed via glutamine or the urea cycle.

Ammonium ions are produced during amino acid degradation and blood concentration is typically low due to their toxicity. Ammonia is toxic to cells as it reduces TCA cycle activity, affects neurotransmitter synthesis and creates an alkaline pH. Detoxification occurs in two steps, firstly ammonia is used to synthesise glutamine when combined with glutamate. Glutamine can then be used to synthesise nitrogen compounds such as purines and pyramidines.

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It is either then transported to the kidney, where the ammonia is directly excreted, or to the liver where it is used to make urea. The urea can then also be transported to the kidneys where the ammonia can be directly excreted in urine. The liver is important in the metabolic activation of Vitamin D.

It is carried to the liver in the blood where it is first converted to the prohormone calcifediol via hydroxylation. This calcifediol is then transported to the kidneys where it is converted into calcitriol, the biologically active form of Vitamin D. The conversion of calcifediol to calcitriol is catalysed by hydroxyvitamin D3 1-alpha-hydroxylase. This conversion is stimulated by parathyroid hormone and low calcium.

Hyperammonaemia is a metabolic disturbance in which there is an excess of ammonia in the blood. It can be caused by a variety of things, both congenital and acquired:. It is potentially a very dangerous condition due to the effects of ammonia on the body and patients often present with vomiting, ataxia, lethargy, weakness, confusion and tachypnoea.