Phospholipid Scramblase 1 Controls Efficient Neurotransmission and Synaptic Vesicle Retrieval at Cerebellar Synapses.

Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission.

Restoring cellular copper homeostasis in Alzheimer disease: a novel peptide shuttle is internalized by an ATP-dependent endocytosis pathway involving Rab5- and Rab14-endosomes.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects.

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid.

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids.

Development of Cu(ii)-specific peptide shuttles capable of preventing Cu-amyloid beta toxicity and importing bioavailable Cu into cells.

Copper (Cu) in its ionic forms is an essential element for mammals and its homeostasis is tightly controlled. Accordingly, Cu-dyshomeostasis can be lethal as is the case in the well-established genetic Wilson's and Menkes diseases. In Alzheimer's disease (AD), Cu-accumulation occurs in amyloid plaques, where it is bound to the amyloid-beta peptide (Aβ).

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion.

Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions.

Phospholipase D1-generated phosphatidic acid modulates secretory granule trafficking from biogenesis to compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis.

Catestatin in innate immunity and Cateslytin-derived peptides against superbugs.

Chromogranin A (CgA) is the precursor of several antimicrobial peptides, such as Catestatin (Cts, bovine CgA344-364), initially described as a potent inhibitor of catecholamines. This peptide displays direct antimicrobial activities and contributes to immune system regulation. The aim of the present study is to investigate a designed peptide based on Cts to fight infections against superbugs and more particularly Staphylococcus aureus.

Protocol for electron microscopy ultrastructural localization of the fusogenic lipid phosphatidic acid on plasma membrane sheets from chromaffin cells.

The glycerophospholipid phosphatidic acid (PA) is a key player in regulated exocytosis, but little is known about its localization at the plasma membrane. Here, we provide a protocol for precisely determining the spatial distribution of PA at exocytotic sites by electron microscopy. Using primary bovine chromaffin cells expressing a PA sensor (Spo20p-GFP), we describe the process for cell stimulation and detergent-free preparation of plasma membrane sheets.

Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells.

Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion.

Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements.

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium.

Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells.

Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr is critical for exocytosis. Considering that Tyr phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells.

Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA.

Phosphatidic acid metabolism regulates neuroendocrine secretion but is not under the direct control of lipins.

Phosphatidic acid (PA) produced by phospholipase D1 has been shown to contribute to secretory vesicle exocytosis in a large number of cell models. Among various hypotheses, PA may contribute to recruit and/or activate at the exocytotic site a set of proteins from the molecular machinery dedicated to secretion, but also directly influence membrane curvature thereby favoring membrane rearrangements required for membrane fusion. The release of informative molecules by regulated exocytosis is a tightly controlled process.

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells.

Annexin A2 (AnxA2) is a calcium and lipid binding protein involved in neuroendocrine secretion. We have previously demonstrated that AnxA2 participates in the formation and/or stabilization of lipid microdomains required for structural and spatial organization of the exocytotic machinery in chromaffin cells. However, the regulation of AnxA2 is not fully understood.

Annexin A2, an essential partner of the exocytotic process in chromaffin cells.

Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, such as neuroendocrine cells. In chromaffin cells, cytosolic annexin A2 is recruited to the plasma membrane upon cell stimulation. Here, we review the latest evidence detailing the functional importance of annexin A2 in different stages of exocytosis.

Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery.

Annexin A2-dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis.

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells.

Exocytosis and endocytosis in neuroendocrine cells: inseparable membranes!

Although 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 neurotransmitters, 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.

Lipids in Regulated Exocytosis: What are They Doing?

The regulated secretory pathway in neuroendocrine cells ends with the release of hormones and neurotransmitters following a rise in cytosolic calcium. This process known as regulated exocytosis involves the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the synaptic vesicle VAMP (synaptobrevin), and the plasma membrane proteins syntaxin and SNAP-25. Although there is much evidence suggesting that SNARE proteins play a key role in the fusion machinery, other cellular elements regulating the kinetics, the extent of fusion, and the preparation of vesicle for release have received less attention.

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.

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.

Lipid dynamics in exocytosis.

Regulated exocytosis of neurotransmitter- and hormone-containing vesicles underpins neuronal and hormonal communication and relies on a well-orchestrated series of molecular interactions. This in part involves the upstream formation of a complex of SNAREs and associated proteins leading to the eventual fusion of the vesicle membrane with the plasma membrane, a process that enables content release. Although the role of lipids in exocytosis is intuitive, it has long been overlooked at least compared to the extensive work on SNAREs.