Vesicle Maturation Research Articles

Dynamic structure of membrane-anchored Arf•GTP

Arfs (ADP ribosylation factors) are N-myristoylated GTP/GDP switch proteins playing key regulatory roles in vesicle transport in eukaryotic cells. ARFs execute their roles by anchoring to membrane surfaces where they interact with other proteins to initiate budding and maturation of transport vesicles. However, existing structures of Arf•GTP are limited to non-myristoylated and truncated forms with impaired membrane binding. We report a high resolution NMR structure for full-length myristoylated yeast (Saccharomyces cerevisiae) Arf1 in complex with a membrane mimic. The two domain structure, in which the myristoylated N-terminal helix is separated from the C-terminal domain by a flexible linker, suggests a level of adaptability in binding modes for the myriad of proteins with which Arf interacts, and allows predictions of specific lipid binding sites on some of these proteins.

Focus on the Spatial Organization of Signalling

Since the early days of light microscopy it has been apparent that cells are highly organized and compartmentalized. However, only in recent decades has the molecular basis of this spatial organization and its functional role in cellular signalling events emerged. Furthermore, modern molecular and imaging techniques, together with the intricate combination of cellular and developmental biological approaches, are extending our understanding of how individual cells respond to their environment and are organized within a tissue. Years of molecular and genetic analysis, coupled with recent advances in large‐scale approaches, may have identified many of the core components of signalling pathways, although new layers of regulation, such as small RNAs and alternative splicing, continue to add complexity. Although the parts list may soon be completed, our understanding at the level of signal integration and cross‐talk remains limited. The key to understanding cellular regulation is to complement biochemical assays in vitro with analysis on how spatial organization contributes to the properties of intra‐ and intercellular communication. This requires a real‐time quantitative analysis of endogenous components in the cellular context, which has only recently became possible with advanced microscopy techniques and biosensors. These developments open a new era in the analysis of spatial organization on different biological scales from molecules to cells to tissues and organs. The EMBO Journal covers a broad spectrum of subject areas and approaches, ranging from biochemical to cell biological, as well as those at the organism level. However, the journal has a longstanding tradition of hosting important papers on signalling, which …

1Alpha,25-(OH)2D3 stimulates matrix vesicle maturation during early osteoblast differentiation leading to enhanced extracellular matrix mineralization during late differentiation

1Alpha,25-(OH)2D3 stimulates matrix vesicle maturation during early osteoblast differentiation leading to enhanced extracellular matrix mineralization during late differentiation

Phosphatidylinositol 4-Phosphate Controls Both Membrane Recruitment and a Regulatory Switch of the Rab GEF Sec2p

Sec2p is the guanine nucleotide exchange factor (GEF) that activates the Rab GTPase Sec4p on secretory vesicles. Sec2p also binds a Rab acting earlier in the secretory pathway, Ypt32-GTP, forming a Rab GEF cascade. Ypt32p and the Sec4p effector Sec15p (a component of the exocyst complex) compete for binding to Sec2p. Indeed Ypt32p initially recruits Sec2p, but subsequently allows a handoff of active Sec2p/Sec4p to Sec15p. Intriguingly, Golgi-associated phosphatidylinositol 4-phosphate (PI4P) works together with Ypt32-GTP in this context. PI4P inhibits Sec2p-Sec15p interactions, promoting recruitment of Sec2p by Ypt32p as secretory vesicles form. However, PI4P levels appear to decline as vesicles reach secretory sites, allowing Sec15p to replace Ypt32p as vesicles mature. In this way, the regulation of PI4P levels may switch Sec2p/Sec4p function during vesicle maturation, from a Rab GEF recruitment cascade involving Ypt32p to an effector positive feedback loop involving Sec15p.

The yeast dynamin-like protein Vps1: vps1 mutations perturb the internalization and the motility of endocytic vesicles and endosomes via disorganization of the actin cytoskeleton

Mammalian dynamin is responsible for scission of endocytic vesicles from the plasma membrane. A previous study showed that Vps1, a yeast dynamin-like protein, plays an important role in pheromone receptor internalization ( Yu and Cai, 2004; J. Cell Sci. 117, 3839–3853). However, the details of how Vps1 acts in various phases of endocytosis including early internalization of the endocytic vesicle are poorly understood. To investigate the potential roles of Vps1 in both endocytic vesicle formation/maturation on the plasma membrane and endocytic vesicle internalization, time-lapse fluorescent images of GFP-tagged endocytic markers in live cells were analyzed using a particle tracking software. The loss of Vps1 leads to a robust increase in the lifespan of newly forming cortical endocytic vesicles carrying Las17-GFP, Ede1-GFP, Sla1-GFP, and Abp1-GFP, indicating that Vps1 is required for the proper assembly and maturation of endocytic vesicles. Particle track analysis revealed that Abp1-GFP vesicles in vps1 null cells moved a relatively short distance away from the cell membrane due to their non-directional movement. Furthermore, we found that the GTPase and the GED domains of Vps1 are required for the proper endocytic function of Vps1. Our tracking analysis data also revealed that the post-internalized vesicle motility en route to the vacuole was decreased significantly, perhaps due to severe disruption of the actin cables in Vps1 mutant cells.

The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes

The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

APP Anterograde Transport Requires Rab3A GTPase Activity for Assembly of the Transport Vesicle

The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by beta- and gamma-secretases leading to accumulation of beta-amyloid (Abeta) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by alpha-secretases prevents Abeta generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by alpha-secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.

Optimization of Immunohistochemical and Fluorescent Antibody Techniques for Localization of Foot-and-Mouth Disease Virus in Animal Tissues

Immunohistochemical (IHC) and fluorescent antibody (FA) techniques were optimized for the detection of Foot-and-mouth disease virus (FMDV) structural and nonstructural proteins in frozen and paraformaldehyde-fixed, paraffin-embedded (PFPE) tissues of bovine and porcine origin. Immunohistochemical localization of FMDV was compared with 7 detection systems, 8 primary antibodies, and 11 epitope retrieval techniques. All serotypes tested (O, A, Asia, C [cryosection]; O, A, Asia [PFPE]) were localized in association with mature vesicles. Multi-label FA was used in conjunction with IHC and conventional histopathology to characterize vesicle maturation in 4 steers and 2 pigs experimentally infected with FMDV. At the edge of advancing vesicles, a consistent finding was acantholytic degeneration of basal keratinocytes surrounding dermal papillae with suprabasilar clefts and microvesiculation. Progression of microvesiculation led to coalescence with the expanding vesicle. Cells at the leading edge of vesicles were positive for FMDV antigens by IHC and FA. Cell marker profile of these cells by FA was consistent with keratinocytes (i.e., cytokeratin [CK]-positive, S100-negative, MHC-II-negative). In rare instances, CK-negative, MHC-II- positive, and FMDV-positive cells (presumptive dendritic cells or macrophages) were identified within dermis subjacent to vesicles.

Direct Visualization of Vesicle Maturation and Plasma Membrane Protein Trafficking

Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET's presence within these structures, and monitor RET's recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.

Rab2 directs a stop-loss program

Small GTPase supports dense core vesicle maturation in worm neurons.

UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation inCaenorhabditis elegans

Small guanosine triphosphatases of the Rab family regulate intracellular vesicular trafficking. Rab2 is highly expressed in the nervous system, yet its function in neurons is unknown. In Caenorhabditis elegans, unc-108/rab-2 mutants have been isolated based on their locomotory defects. We show that the locomotion defects of rab-2 mutants are not caused by defects in synaptic vesicle release but by defects in dense core vesicle (DCV) signaling. DCVs in rab-2 mutants are often enlarged and heterogeneous in size; however, their number and distribution are not affected. This implicates Rab2 in the biogenesis of DCVs at the Golgi complex. We demonstrate that Rab2 is required to prevent DCV cargo from inappropriately entering late endosomal compartments during DCV maturation. Finally, we show that RIC-19, the C. elegans orthologue of the human diabetes autoantigen ICA69, is also involved in DCV maturation and is recruited to Golgi membranes by activated RAB-2. Thus, we propose that RAB-2 and its effector RIC-19 are required for neuronal DCV maturation.

FAPP2 gene downregulation increases tumor cell sensitivity to Fas-induced apoptosis

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts ( Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.

Interplay between Rab5 and PtdIns(4,5)P2 controls early endocytosis in theDrosophilagermline

Phosphoinositides have emerged as key regulators of membrane traffic through their control of the localization and activity of several effector proteins. Both Rab5 and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] are involved in the early steps of the clathrin-dependent endocytic pathway, but little is known about how their functions are coordinated. We have studied the role of PtdIns(4,5)P(2) and Rab5 in the Drosophila germline during oogenesis. We found that Rab5 is required for the maturation of early endocytic vesicles. We show that PtdIns(4,5)P(2) is required for endocytic-vesicle formation, for Rab5 recruitment to endosomes and, consistently, for endocytosis. Furthermore, we reveal a previously undescribed role of Rab5 in releasing PtdIns(4,5)P(2), PtdIns(4,5)P(2)-binding budding factors and F-actin from early endocytic vesicles. Finally, we show that overexpressing the PtdIns(4,5)P(2)-synthesizing enzyme Skittles leads to an endocytic defect that is similar to that seen in rab5 loss-of-function mutants. Hence, our results argue strongly in favor of the hypothesis that the Rab5-dependant release of PtdIns(4,5)P(2) from endosomes that we discovered in this study is crucial for endocytosis to proceed.

Role of Rab27 in synaptic transmission at the squid giant synapse

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Legionella Eukaryotic-Like Type IV Substrates Interfere with Organelle Trafficking

Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.

Ablation of GalNAc-4-sulfotransferase-1 enhances reproduction by altering the carbohydrate structures of luteinizing hormone in mice

Luteinizing hormone (LH), produced in the anterior lobe of the pituitary, is a member of the hypothalamic-pituitary-gonad axis that is required for production of the sex hormones estradiol, progesterone, and testosterone. Perturbations in levels of hormones associated with this axis can result in defects in sexual development and maturity. LH bears unique N-linked carbohydrate units that terminate with a sulfated N-acetylgalactosamine structure (GalNAc-4-SO(4)) that mediates its clearance from the blood. To determine the significance of this terminal structure, we ablated the gene encoding the sulfotransferase responsible for sulfate addition to GalNAc on LH, GalNAc-4-sulfotransferase-1 (GalNAc-4-ST1) in mice. Mice lacking GalNAc-4-ST1 exhibited increased levels of circulating LH. In male mice, this resulted in elevated levels of testosterone and precocious maturation of testis and seminal vesicles. Female mice lacking GalNAc-4-ST1 demonstrated elevated estrogen levels and exhibited precocious sexual maturation and increased fecundity. Female mice remained in estrus for prolonged periods and produced almost 50% more litters per mouse than wild-type mice over the same period of time. Thus, sulfate modification of the terminal glycosylation of LH plays a central role in regulating the hypothalamic-pituitary-gonad axis in vivo.

Vesicle Priming and Recruitment by ubMunc13-2 Are Differentially Regulated by Calcium and Calmodulin

Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.

Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum

The intra-erythrocytic stages of the malaria parasite endocytose large quantities of the surrounding erythrocyte cytoplasm and deliver it to a digestive food vacuole via endocytic vesicles. Digestion provides amino acids for parasite protein synthesis and is required to maintain the osmotic integrity of the host cell. The parasite endocytic pathway has been described morphologically by electron microscopy, but the molecular mechanisms that mediate and regulate it remain elusive. Given the involvement of actin in endocytosis in other eukaryotes, we have used actin inhibitors to assess the requirement for this protein in the endocytic pathway of the human malaria parasite, Plasmodium falciparum. Treatment of cultures with cytochalasin D did not affect haemoglobin levels in the parasites when co-administered with protease inhibitors, and neither did it affect the uptake of the endocytic tracer horseradish peroxidase, suggesting the absence of actin in the mechanism of endocytosis. However, in the absence of protease inhibitors, treated parasites contained increased levels of haemoglobin due to an accumulation of enlarged endocytic vesicles, as determined by immunofluorescence and electron microscopy, suggesting a role for actin in vesicle trafficking, possibly by mediating vesicle maturation and/or fusion to the digestive vacuole. In contrast to cytochalasin D, treatment with jasplakinolide led to an inhibition of endocytosis, an accumulation of vesicles closer to the plasma membrane and a marked concentration of actin in the parasite cortex. We propose that the stabilization of cortical actin filaments by jasplakinolide interferes with normal endocytic vesicle formation and migration from the cell periphery.

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.