Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation.

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis.

The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.

Directional mast cell degranulation of tumor necrosis factor into blood vessels primes neutrophil extravasation.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster.

Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. Here, we present evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. We propose that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process.

Synaptic vesicle distribution by conveyor belt.

The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution.

Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture.

Neurotransmission requires anterograde axonal transport of dense core vesicles (DCVs) containing neuropeptides and active zone components from the soma to nerve terminals. However, it is puzzling how one-way traffic could uniformly supply sequential release sites called en passant boutons. Here, Drosophila neuropeptide-containing DCVs are tracked in vivo for minutes with a new method called simultaneous photobleaching and imaging (SPAIM). Surprisingly, anterograde DCVs typically bypass proximal boutons to accumulate initially in the most distal bouton. Then, excess distal DCVs undergo dynactin-dependent retrograde transport back through proximal boutons into the axon. Just before re-entering the soma, DCVs again reverse for another round of anterograde axonal transport. While circulating over long distances, both anterograde and retrograde DCVs are captured sporadically in en passant boutons. Therefore, vesicle circulation, which includes long-range retrograde transport and inefficient bidirectional capture, overcomes the limitations of one-way anterograde transport to uniformly supply release sites with DCVs.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

Cell wall dynamics stabilize tip growth in a filamentous fungus.

Hyphal tip growth allows filamentous fungi to colonize space, reproduce, or infect. It features remarkable morphogenetic plasticity including unusually fast elongation rates, tip turning, branching, or bulging. These shape changes are all driven from the expansion of a protective cell wall (CW) secreted from apical pools of exocytic vesicles. How CW secretion, remodeling, and deformation are modulated in concert to support rapid tip growth and morphogenesis while ensuring surface integrity remains poorly understood. We implemented subresolution imaging to map the dynamics of CW thickness and secretory vesicles in Aspergillus nidulans. We found that tip growth is associated with balanced rates of CW secretion and expansion, which limit temporal fluctuations in CW thickness, elongation speed, and vesicle amount, to less than 10% to 20%. Affecting this balance through modulations of growth or trafficking yield to near-immediate changes in CW thickness, mechanics, and shape. We developed a model with mechanical feedback that accounts for steady states of hyphal growth as well as rapid adaptation of CW mechanics and vesicle recruitment to different perturbations. These data provide unprecedented details on how CW dynamics emerges from material secretion and expansion, to stabilize fungal tip growth as well as promote its morphogenetic plasticity.

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

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.

Swip-1 promotes exocytosis of glue granules in the exocrine Drosophila salivary gland.

Exocytosis is a fundamental cellular process by which cells secrete cargos from their apical membrane into the extracellular lumen. Cargo release proceeds in sequential steps that depend on coordinated assembly and organization of an actin cytoskeletal network. Here, we identified the conserved actin-crosslinking protein Swip-1 as a novel regulator controlling exocytosis of glue granules in the Drosophila salivary gland. Real-time imaging revealed that Swip-1 is simultaneously recruited with F-actin onto secreting granules in proximity to the apical membrane. We observed that Swip-1 is rapidly cleared at the point of secretory vesicle fusion and colocalizes with actomyosin network around the fused vesicles. Loss of Swip-1 function impairs secretory cargo expulsion, resulting in strongly delayed secretion. Thus, our results uncover a novel role of Swip-1 in secretory vesicle compression and expulsion of cargo during regulated exocytosis. Remarkably, this function neither requires Ca2+ binding nor dimerization of Swip-1. Our data rather suggest that Swip-1 regulates actomyosin activity upstream of Rho-GTPase signaling to drive proper vesicle membrane crumpling and expulsion of cargo.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Regulated Restructuring of Mucins During Secretory Granule Maturation In Vivo.

Mucins are large, highly glycosylated transmembrane and secreted proteins that line and protect epithelial surfaces. However, the details of mucin biosynthesis and packaging in vivo are largely unknown. Here, we demonstrate that multiple distinct mucins undergo intragranular restructuring during secretory granule maturation in vivo, forming unique structures that are spatially segregated within the same granule. We further identify temporally-regulated genes that influence mucin restructuring, including those controlling pH (Vha16-1), Ca2+ ions (fwe) and Cl- ions (Clic and ClC-c). Finally, we show that altered mucin glycosylation influences the dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular segregation of mucins through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct mucins are efficiently packaged into and secreted from secretory granules may provide insight into diseases resulting from defects in mucin secretion.

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.

Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells.

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila.

Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 µm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.

Distribution of membrane trafficking system components across ciliate diversity highlights heterogenous organelle-associated machinery.

The ciliate phylum is a group of protists noted for their unusual membrane trafficking system and apparent environmental ubiquity; as highly successful microbial predators, they are found in all manner of environments and the ability for specific species to adapt to extremely challenging conditions makes them valued as bioindicators. Ciliates have also been used for many years as cell biological models because of their large cell size and ease of culturing, and for many fundamental cell structures, particularly membrane-bound organelles, ciliates were some of the earliest organisms in which these were observed via microscopy. In this study, we carried out a comparative genomic survey of selected membrane trafficking proteins in a pan-ciliate transcriptome and genome dataset. We observed considerable loss of membrane trafficking system (MTS) proteins that would indicate a loss of machinery that is generally conserved across eukaryotic diversity, even after controlling for potentially incomplete genome representation. In particular, the complete DSL1 complex was missing in all surveyed ciliates. This protein complex has been shown as involved in peroxisome biogenesis in some model systems, and a paucity of DSL1 components has been indicative of degenerate peroxisome. However, Tetrahymena thermophila (formerly Tetrahymena pyroformis) was one of the original models for visualizing peroxisomes. Conversely, the AP3 complex essential for mucocyst maturation in T. thermophila, is poorly conserved despite the presence of secretory lysosome-related organelles across ciliate diversity. We discuss potential resolutions for these apparent paradoxes in the context of the heterogenous distribution of MTS machinery across the diversity of ciliates.