Formation Of Secretory Vesicles Research Articles

The role of crinophagy in quality control of the regulated secretory pathway

In specialized secretory cells that produce and release biologically active substances in a regulated fashion, tight control of both the quantity and quality of secretory material is of paramount importance. During crinophagy, abnormal, excess or obsolete secretory granules directly fuse with lysosomes to yield crinosomes, in which the delivered secretory material is degraded. Crinophagy maintains the proper intracellular pool of secretory granules, and it is enhanced when secretory material accumulates because of compromised secretion. Recent studies highlight that it can even degrade newly formed, nascent secretory granules that shed from the trans-Golgi network. This implies that crinophagy provides a quality control checkpoint acting at the formation of secretory vesicles, and this degradation mechanism might survey secretory granules throughout their maturation. Of note, a plethora of human disorders is associated with defective lysosomal clearance of secretory material via crinophagy or similar pathways, including macro- or micro-autophagic degradation of secretory granules (referred to here as macro- and micro-secretophagy, respectively). In our Review, we summarize key recent advances in this field and discuss potential links with disease.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

Structural basis for specific activation of the Rab11 GTPase by the TRAPPII complex

Secretion is an essential cellular function in which proteins and lipids are transported from the Golgi to the plasma membrane via secretory vesicles. The formation of secretory vesicles is regulated by activation of the Rab GTPase Rab11. The activation of Rab GTPases at membrane compartments is dependent on the localization of their specific Guanosine Exchange Factors (GEFs), which catalyze the exchange of GDP with GTP and stabilize the active GTPase on the membrane. The multisubunit TRAPPII complex acts as Rab11 GEF and recruits it to the trans‐Golgi network (TGN) membrane. More than 60 different Rabs are present in mammals and budding yeast has 11 different Rabs. Specific localization of each of these Rabs to different membrane compartments is important for determination of organelle identity and membrane organization of these compartments. While multiple GTPases and GEFs localize at the TGN, the mechanistic details of how TRAPPII specifically activates Rab11, and not other GTPases, is unknown. Moreover, another related GEF, the TRAPPIII complex, shares the same catalytic subunits with TRAPPII and yet activates another GTPase, Rab1. A steric gating mechanism has been proposed in which TRAPPII selects against Rab1 based on the length of the C‐terminal hypervariable domain. We determined the structure of yeast TRAPPII bound to its substrate Rab11 using cryo‐EM at an overall resolution of 3.7Å. Analysis of the atomic structure has revealed specific interactions between Rab11 and the TRAPPII complex, and how TRAPPII interacts with the membrane surface. We tested the physiological relevance of these observed interactions using in vivo functional studies and in vitro reconstitution of nucleotide exchange. We show that the TRAPPII subunit Trs130 provides a ‘leg’ which lifts the TRAPP catalytic site above the membrane, thereby preventing access to the catalytic site by Rab1. TRAPPIII complex selects against Rab11 based on repulsive interactions with the catalytic subunits. We also show an alternative conformation of the TRAPPII complex which may facilitate access of Rab11 to the TRAPP catalytic site. Taken together, these experiments reveal the mechanism of specific activation of Rab11 on the membrane by TRAPPII, a key step for initiation of secretory vesicle formation. As Rab11 and the TRAPPII complex are conserved throughout eukaryotes, this mechanism may be widely conserved across all eukaryotic secretory systems.

Arf1 Directly Recruits the Pik1‐Frq1 PI4K Complex to Regulate the Final Stages of Golgi Maturation

Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PtdIns4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIβ) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PtdIns4P production, but whether Pik1 is a direct Arf1 effector has not been established. Using a combination of live-cell time-lapse imaging analyses, acute PtdIns4P depletion experiments, and in vitro protein-protein interaction assays on Golgi-mimetic membranes, we have found that Arf1 initiates the final stages of Golgi maturation by tightly controlling PtdIns4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PtdIns4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work, along with ongoing structural investigations, establishes the regulatory and temporal context surrounding Golgi PtdIns4P production and its precise roles in Golgi maturation.

Enhancement in Phospholipase D Activity as a New Proposed Molecular Mechanism of Haloperidol-Induced Neurotoxicity.

Membrane phospholipase D (PLD) is associated with numerous neuronal functions, such as axonal growth, synaptogenesis, formation of secretory vesicles, neurodegeneration, and apoptosis. PLD acts mainly on phosphatidylcholine, from which phosphatidic acid (PA) and choline are formed. In turn, PA is a key element of the PLD-dependent secondary messenger system. Changes in PLD activity are associated with the mechanism of action of olanzapine, an atypical antipsychotic. The aim of the present study was to assess the effect of short-term administration of the first-generation antipsychotic drugs haloperidol, chlorpromazine, and fluphenazine on membrane PLD activity in the rat brain. Animals were sacrificed for a time equal to the half-life of the antipsychotic drug in the brain, then the membranes in which PLD activity was determined were isolated from the tissue. The results indicate that only haloperidol in a higher dose increases the activity of phospholipase D. Such a mechanism of action of haloperidol has not been described previously. Induction of PLD activity by haloperidol may be related to its mechanism of cytotoxicity. The finding could justify the use of PLD inhibitors as protective drugs against the cytotoxicity of first-generation antipsychotic drugs like haloperidol.

Phagocytosis of monosodium urate crystals by human synoviocytes induces inflammation.

Gout is distinguished by an inflammatory process that is mediated by phagocytosis of monosodium urate (MSU) crystals in synoviocytes by regulation of unknown mechanisms. Here we suggest that the synovial cells play a crucial role in gouty arthritis by activating inflammation by MSU uptake and increasing the secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNF-α, MCP-1, and the growth factors NGF and HGF. We discuss some co-existing features in synoviocytes, including anomalous morphologies of the cells, and microvesicle formation, dysregulation in VEGF gene expression. We provide evidence that phagocytosis of MSU crystals triggers an inflammatory cellular state in synoviocytes in the pathogenesis of crystal-induced arthritis.

Ride the wave: Retrograde trafficking becomes Ca2+ dependent with BAIAP3.

The functions of four of the five proteins in the mammalian uncoordinated-13 (Munc13) family have been identified as priming factors in SNARE-dependent exocytosis. In this issue, Zhang et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201702099) show that the fifth member, BAIAP3 (brain-specific angiogenesis inhibitor I-associated protein 3), acts in retrograde trafficking by returning secretory vesicle material to the trans-Golgi network. In its absence, secretory vesicle formation is impaired, leading to accumulation of immature vesicles, or lysosomal vesicle degradation.

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl- fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis.

Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.

A Wntless–SEC12 complex on the ER membrane regulates early Wnt secretory vesicle assembly and mature ligand export

Wntless (Wls) transports Wnt molecules for secretion; however, the cellular mechanism underlying the initial assembly of Wnt secretory vesicles is still not fully defined. Here, we performed proteomic and mutagenic analyses of mammalian Wls, and report a mechanism for formation of early Wnt secretory vesicles on ER membrane. Wls forms a complex with SEC12 (also known as PREB), an ER membrane-localized guanine nucleotide-exchange factor (GEF) activator of the SAR1 (the SAR1A isoform) small GTPase. Compared to palmitoylation-deficient Wnt molecules, binding of mature Wnt to Wls increases Wls-SEC12 interaction and promotes association of Wls with SAR1, the key activator of the COPII machinery. Incorporation of Wls into this exporting ER compartment is affected by Wnt ligand binding and SEC12 binding to Wls, as well as the structural integrity and, potentially, the folding of the cytosolic tail of Wls. In contrast, Wls-SEC12 binding is stable, with the interacting interface biochemically mapped to cytosolic segments of individual proteins. Mutant Wls that fails to communicate with the COPII machinery cannot effectively support Wnt secretion. These data suggest that formation of early Wnt secretory vesicles is carefully regulated to ensure proper export of functional ligands.

Real-time insights into regulated exocytosis.

Real-time imaging of regulated exocytosis in secreting organs can provide unprecedented temporal and spatial detail. Here, we highlight recent advances in 3D time-lapse imaging in Drosophila salivary glands at single-granule resolution. Using fluorescently labeled proteins expressed in the fly, it is now possible to image the dynamics of vesicle biogenesis and the cytoskeletal factors involved in secretion. 3D imaging over time allows one to visualize and define the temporal sequence of events, including clearance of cortical actin, fusion pore formation, mixing of the vesicular and plasma membranes and recruitment of components of the cytoskeleton. We will also discuss the genetic tools available in the fly that allow one to interrogate the essential factors involved in secretory vesicle formation, cargo secretion and the ultimate integration of the vesicular and plasma membranes. We argue that the combination of high-resolution real-time imaging and powerful genetics provides a platform to investigate the role of any factor in regulated secretion.

Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

Journey to the cell surface--the central role of the trans-Golgi network in plants.

The secretion of proteins, lipids, and carbohydrates to the cell surface is essential for plant development and adaptation. Secreted substances synthesized at the endoplasmic reticulum pass through the Golgi apparatus and trans-Golgi network (TGN) en route to the plasma membrane via the conventional secretion pathway. The TGN is morphologically and functionally distinct from the Golgi apparatus. The TGN is located at the crossroads of many trafficking pathways and regulates a range of crucial processes including secretion to the cell surface, transport to the vacuole, and the reception of endocytic cargo. This review outlines the TGN's central role in cargo secretion, showing that its behavior is more complex and controlled than the bulk-flow hypothesis suggests. Its formation, structure, and maintenance are discussed along with the formation and release of secretory vesicles.

Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

O-glycosylation regulates polarized secretion by modulating Tango1 stability.

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.

Mature VLDL triggers the biogenesis of a distinct vesicle from the trans-Golgi network for its export to the plasma membrane

Post-Golgi trafficking of mature VLDL (very-low-density lipoprotein) is crucial in maintaining normal TAG (triacylglycerol) homoeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the TGN (trans-Golgi network) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich PG-VTVs (post-TGN VLDL transport vesicles) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions, whereas protein transport vesicles were present in relatively higher-density fractions of the same sucrose gradient. EM revealed large intact PG-VTVs ranging 300-350 nm in size. The biogenesis of PG-VTVs from the TGN required cytosol, ATP, GTP hydrolysis and incubation at 37°C. PG-VTVs concentrated the VLDL proteins: apolipoproteins apoB100, apoAIV, apoAI and apoE, but did not contain either albumin or transferrin. Proteinase K treatment did not degrade VLDL core proteins, suggesting that PG-VTVs were sealed. PG-VTVs were able to fuse with and deliver VLDL to the PM (plasma membrane) in a vectorial manner. We conclude that we have identified a new TGN-derived vesicle, the PG-VTV, which specifically transports mature VLDL from the TGN to the PM.

Targeting and Regulation of Cell Wall Synthesis During Tip Growth in Plants

Root hairs and pollen tubes are formed through tip growth, a process requiring synthesis of new cell wall material and the precise targeting and integration of these components to a selected apical plasma membrane domain in the growing tips of these cells. Presence of a tip-focused calcium gradient, control of actin cytoskeleton dynamics, and formation and targeting of secretory vesicles are essential to tip growth. Similar to cells undergoing diffuse growth, cellulose, hemicelluloses, and pectins are also deposited in the growing apices of tip-growing cells. However, differences in the manner in which these cell wall components are targeted and inserted in the expanding portion of tip-growing cells is reflected by the identification of elements of the plant cell wall synthesis machinery which have been shown to play unique roles in tip-growing cells. In this review, we summarize our current understanding of the tip growth process, with a particular focus on the subcellular targeting of newly synthesized cell wall components, and their roles in this form of plant cell expansion.

Selenoneine, a novel selenium-containing compound, mediates detoxification mechanisms against methylmercury accumulation and toxicity in zebrafish embryo.

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N α,N α,N α-trimethyl-l-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1). Selenoneine was incorporated into human embryonic kidney HEK293 cells transiently overexpressing OCTN1 and zebrafish blood cells by OCTN1. The K m for selenoneine uptake was 13.0 μM in OCTN1-overexpressing HEK293 cells and 9.5 μM in zebrafish blood cells, indicating high affinity of OCTN1 for selenoneine in human and zebrafish cells. When such OCTN1-expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg were enhanced by selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1-deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Furthermore, Hg accumulation was decreased in OCTN1-overexpressing HEK293 cells, but not in mock vector-transfected cells, indicating that selenoneine and OCTN1 can regulate MeHg detoxification in human cells. Thus, the selenoneine-mediated OCTN1 system regulates secretory lysosomal vesicle formation and MeHg demethylation.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-013-9508-1) contains supplementary material, which is available to authorized users.

A Pair of Crescent-shaped Proteins Shape Vesicles at the Golgi

The Golgi network may look like a stack of pancakes, but it functions like a high-speed, just-in-time cargo facility, modifying, sorting, and packaging proteins for destinations within and beyond the cell. Among these proteins are hormones, such as insulin and growth hormone, that are shuttled into secretory vesicles, where they remain until the cell receives the call to ship them out. The molecular dynamics of those secretory vesicles are becoming better known, thanks to two new studies that identify a pair of interacting proteins–PICK1 and ICA69—as critical players in the formation and maturation of secretory vesicles, with potential roles in endocrine diseases.

Strong antioxidant selenoneine in tuna blood: its roles in selenium redox metabolism

Selenium‐containing imidazole compound, 2‐selenyl‐Nα, Nα, Nα‐ trimethyl‐L‐histidine, “selenoneine” in tuna blood had a strong antioxidant capacity and bound to hemoglobin and myoglobin (Mb), to protect them from iron autoxidation, and reacted with radicals and methylmercury (MeHg). Cell growth of human and zebrafish cultured cells were enhanced in the presence of selenoneine at 5–100 nM, and GPx1 gene expression was induced in dose‐dependent manner. Administration of selenoneine to yellowtail fish reduced ROS and met‐Mb formation, and the meat color change in dark muscle was protected. Selenoneine was incorporated into human embryonic kidney HEK293 transiently overexpressed with organic cations/carnitine transporter‐1 (OCTN1) and zebrafish embryo cells by OCTN1. The Km value of selenoneine uptake was determined to be 13.0 μM in OCTN1‐oeverexpressed HEK293 cells and 9.5 μM in zebrafish erythrocytes, respectively, indicating that selenoneine is the most specific substrate for the OCTN1 in the human and zebrafish cells. When such OCTN1‐expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg was enhanced by the presence of selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1‐deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Therefore, the selenoneine‐mediated OCTN1 transport system accelerates secretory extracellular lysosomal vesicle formation and thus the demethylation of MeHg.