How important are Rho GTPases in neurosecretion?

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Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore

Exocytosis and Endocytosis in Neuroendocrine Cells: Inseparable Membranes!

International audienceAlthough much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid toward understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotrans-mitters, neuropeptides, and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval

Phospholipid Scramblase-1-Induced Lipid Reorganization Regulates Compensatory Endocytosis in Neuroendocrine Cells

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1−/−mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate.

HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P2-binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P2 perturbs several PtdIns(4,5)P2-dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.journal articleresearch support, non-u.s. gov't2013 Jan 152012 11 23importe

An asymmetric approach to preserve common intervals while sorting by reversals

Dias Vieira Braga M, Gautier C, Sagot M-F. An asymmetric approach to preserve common intervals while sorting by reversals. Algorithms for Molecular Biology. 2009;4(1):16.Background: The reversal distance and optimal sequences of reversals to transform a genome into another are useful tools to analyse evolutionary scenarios. However, the number of sequences is huge and some additional criteria should be used to obtain a more accurate analysis. One strategy is searching for sequences that respect constraints, such as the common intervals (clusters of co-localised genes). Another approach is to explore the whole space of sorting sequences, eventually grouping them into classes of equivalence. Recently both strategies started to be put together, to restrain the space to the sequences that respect constraints. In particular an algorithm has been proposed to list classes whose sorting sequences do not break the common intervals detected between the two inital genomes A and B. This approach may reduce the space of sequences and is symmetric (the result of the analysis sorting A into B can be obtained from the analysis sorting B into A). Results: We propose an alternative approach to restrain the space of sorting sequences, using progressive instead of initial detection of common intervals (the list of common intervals is updated after applying each reversal). This may reduce the space of sequences even more, but is shown to be asymmetric. Conclusions: We suggest that our method may be more realistic when the relation ancestor-descendant between the analysed genomes is clear and we apply it to do a better characterisation of the evolutionary scenario of the bacterium Rickettsia felis with respect to one of its ancestors

Sampling solution traces for the problem of sorting permutations by signed reversals

International audienceBackgroundTraditional algorithms to solve the problem of sorting by signed reversals output just one optimal solution while the space of all optimal solutions can be huge. A so-called trace represents a group of solutions which share the same set of reversals that must be applied to sort the original permutation following a partial ordering. By using traces, we therefore can represent the set of optimal solutions in a more compact way. Algorithms for enumerating the complete set of traces of solutions were developed. However, due to their exponential complexity, their practical use is limited to small permutations. A partial enumeration of traces is a sampling of the complete set of traces and can be an alternative for the study of distinct evolutionary scenarios of big permutations. Ideally, the sampling should be done uniformly from the space of all optimal solutions. This is however conjectured to be ♯P-complete.ResultsWe propose and evaluate three algorithms for producing a sampling of the complete set of traces that instead can be shown in practice to preserve some of the characteristics of the space of all solutions. The first algorithm (RA) performs the construction of traces through a random selection of reversals on the list of optimal 1-sequences. The second algorithm (DFALT) consists in a slight modification of an algorithm that performs the complete enumeration of traces. Finally, the third algorithm (SWA) is based on a sliding window strategy to improve the enumeration of traces. All proposed algorithms were able to enumerate traces for permutations with up to 200 elements.ConclusionsWe analysed the distribution of the enumerated traces with respect to their height and average reversal length. Various works indicate that the reversal length can be an important aspect in genome rearrangements. The algorithms RA and SWA show a tendency to lose traces with high average reversal length. Such traces are however rare, and qualitatively our results show that, for testable-sized permutations, the algorithms DFALT and SWA produce distributions which approximate the reversal length distributions observed with a complete enumeration of the set of traces

PROTEINES G HETEROTRIMERIQUES ET EXOCYTOSE (IDENTIFICATION D'UN MECANISME POTENTIEL DE REGULATION PAR LA PHOSDUCIN-LIKE PROTEIN DANS LES CELLULES CHROMAFFINES)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF