Formation Of Transport Vesicles Research Articles

S1P regulates intervertebral disc aging by mediating endoplasmic reticulum-mitochondrial calcium ion homeostasis.

As the aging process progresses, age-related intervertebral disc degeneration (IVDD) is becoming an emerging public health issue. Site-1 protease (S1P) has recently been found to be associated with abnormal spinal development in patients with mutations and has multiple biological functions. Here, we discovered a reduction of S1P in degenerated and aging intervertebral discs, primarily regulated by DNA methylation. Furthermore, through drug treatment and siRNA-mediated S1P knockdown, nucleus pulposus cells were more prone to exhibit degenerative and aging phenotypes. Conditional KO of S1P in mice resulted in spinal developmental abnormalities and premature aging. Mechanistically, S1P deficiency impeded COP II-mediated transport vesicle formation, which leads to protein retention in the endoplasmic reticulum (ER) and subsequently ER distension. ER distension increased the contact between the ER and mitochondria, disrupting ER-to-mitochondria calcium flow and resulting in mitochondrial dysfunction and energy metabolism disturbance. Finally, using 2-APB to inhibit calcium ion channels and the senolytic drug dasatinib and quercetin (D + Q) partially rescued the aging and degenerative phenotypes caused by S1P deficiency. In conclusion, our findings suggest that S1P is a critical factor in causing IVDD in the process of aging and highlight the potential of targeting S1P as a therapeutic approach for age-related IVDD.

Disordered hinge regions of the AP-3 adaptor complex promote vesicle budding from the late Golgi in yeast.

Vesicles bud from maturing Golgi cisternae in a programmed sequence. Budding is mediated by adaptors that recruit cargoes and facilitate vesicle biogenesis. In Saccharomyces cerevisiae, the AP-3 adaptor complex directs cargoes from the Golgi to the lysosomal vacuole. The AP-3 core consists of small and medium subunits complexed with two non-identical large subunits, β3 (Apl6) and δ (Apl5). The C-termini of β3 and δ were thought to be flexible hinges linking the core to ear domains that bind accessory proteins involved in vesicular transport. We found by computational modeling that the yeast β3 and δ hinges are intrinsically disordered and lack folded ear domains. When either hinge is truncated, AP-3 is recruited to the Golgi, but vesicle budding is impaired and cargoes normally sorted into the AP-3 pathway are mistargeted. This budding deficiency causes AP-3 to accumulate on ring-like Golgi structures adjacent to GGA adaptors that, in wild-type cells, bud vesicles downstream of AP-3 during Golgi maturation. Thus, each of the disordered hinges of yeast AP-3 has a crucial role in mediating transport vesicle formation at the Golgi.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Membrane trafficking functions of the ANTH/ENTH/VHS domain-containing proteins in plants.

Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.

Measuring protein insertion areas in lipid monolayers by fluorescence correlation spectroscopy

Membrane insertion of protein domains is an important step in many membrane remodeling processes, for example, in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. To our knowledge, this is the first optical measurement of this quantity on a system in equilibrium with direct determination of the number of inserted protein and no further assumptions concerning the binding thermodynamics. Whereas macroscopic total area changes in lipid monolayers are typically measured on a Langmuir film balance, finding the number of inserted proteins without perturbing the system and quantitating any small area changes has posed a challenge. Here, we address both issues by performing two-color fluorescence correlation spectroscopy directly on the monolayer. With a fraction of the protein being fluorescently labeled, the number of inserted proteins is determined in situ without resorting to invasive techniques such as collecting the monolayer by aspiration. The second color channel is exploited to monitor a small fraction of labeled lipids to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in COPII reconstitution experiments, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to also be applied to laterally heterogeneous monolayers and bilayers.

Luman/CREB3 knock-down inhibit hCG induced MLTC-1 apoptosis

Luman has been reported to be involved in the formation of COP II-mediated transport vesicles that affect protein transportation and secretion. Western blotting, immunohistochemistry, immunofluorescence, and RT-qPCR indicated that Luman is widely expressed in the male mouse reproductive system. In sperm, Luman was mainly located in the sperm tail, and the expression level increased with sperm maturity. In the testis, Luman was located in Leydig cells. In MLTC-1, a high-concentration hCG treatment significantly increased GRP78, ATF6, p-IRE1, and p-EIF2S1 expression but had no effect on Luman expression. To investigate the role of Luman in hCG-induced ER stress (ERS), experiments were conducted to examine the consequences of short hairpin RNA (shRNA)-mediated Luman knockdown in MLTC-1 cells. Luman knockdown decreased the percentage of S phase cells and up-regulated Cyclin A1, Cyclin B1, and Cyclin D2 expression. ELISA and WB results showed that with Luman knockdown, Cyp11a1, p-IRE1, and p-EIF2S1 expression and testosterone secretion were significantly increased, while GRP78 and CHOP expression were decreased. Flow cytometry results showed that Luman knockdown reduced MLTC-1 cell apoptosis. RT-qPCR and WB results showed that Luman knockdown significantly up-regulated BCL-2 expression and decreased Caspase-3 and BAX expression. These data suggest that Luman is widely expressed in the male mouse reproductive system. In MLTC-1 cells, Luman knockdown up-regulated p-IRE1, p-EIF2S1, and BCL-2 expression and decreased GRP78, CHOP, BAX, and Caspase-3 expression. We propose that Luman knockdown reduces cell apoptosis through the ERS pathway, thereby promoting cell survival and testosterone secretion. These findings provide new insights into the role of Luman in hCG-induced ERS.

The role of AP-4 in cargo export from the trans-Golgi network and hereditary spastic paraplegia.

Heterotetrameric adaptor protein (AP) complexes play key roles in protein sorting and transport vesicle formation in the endomembrane system of eukaryotic cells. One of these complexes, AP-4, was identified over 20 years ago but, up until recently, its function remained unclear. AP-4 associates with the trans-Golgi network (TGN) through interaction with small GTPases of the ARF family and recognizes transmembrane proteins (i.e. cargos) having specific sorting signals in their cytosolic domains. Recent studies identified accessory proteins (tepsin, RUSC2 and the FHF complex) that co-operate with AP-4, and cargos (amyloid precursor protein, ATG9A and SERINC3/5) that are exported from the TGN in an AP-4-dependent manner. Defective export of ATG9A from the TGN in AP-4-deficient cells was shown to reduce ATG9A delivery to pre-autophagosomal structures, impairing autophagosome formation and/or maturation. In addition, mutations in AP-4-subunit genes were found to cause neurological dysfunction in mice and a form of complicated hereditary spastic paraplegia referred to as 'AP-4-deficiency syndrome' in humans. These findings demonstrated that mammalian AP-4 is required for the development and function of the central nervous system, possibly through its role in the sorting of ATG9A for the maintenance of autophagic homeostasis. In this article, we review the properties and functions of AP-4, and discuss how they might explain the clinical features of AP-4 deficiency.

The Small GTPase Superfamily in Plants: A Conserved Regulatory Module with Novel Functions

Small GTP-binding proteins represent a highly conserved signaling module in eukaryotes that regulates diverse cellular processes such as signal transduction, cytoskeletal organization and cell polarity, cell proliferation and differentiation, intracellular membrane trafficking and transport vesicle formation, and nucleocytoplasmic transport. These proteins function as molecular switches that cycle between active and inactive states, and this cycle is linked to GTP binding and hydrolysis. In this review, the roles of the plant complement of small GTP-binding proteins in these cellular processes are described, as well as accessory proteins that control their activity, and current understanding of the functions of individual members of these families in plants-with a focus on the model organism Arabidopsis-is presented. Some potential novel roles of these GTPases in plants, relative to their established roles in yeast and/or animal systems, are also discussed.

Structure of membrane tethers and their role in fusion.

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so-called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.

Clathrin Adaptor Complex-interacting Protein Irc6 Functions through the Conserved C-Terminal Domain

Clathrin coats drive transport vesicle formation from the plasma membrane and in pathways between the trans-Golgi network (TGN) and endosomes. Clathrin adaptors play central roles orchestrating assembly of clathrin coats. The yeast clathrin adaptor-interacting protein Irc6 is an orthologue of human p34, which is mutated in the inherited skin disorder punctate palmoplantar keratoderma type I. Irc6 and p34 bind to clathrin adaptor complexes AP-1 and AP-2 and are members of a conserved family characterized by a two-domain architecture. Irc6 is required for AP-1-dependent transport between the TGN and endosomes in yeast. Here we present evidence that the C-terminal two amino acids of Irc6 are required for AP-1 binding and transport function. Additionally, like the C-terminal domain, the N-terminal domain when overexpressed partially restores AP-1-mediated transport in cells lacking full-length Irc6. These findings support a functional role for Irc6 binding to AP-1. Negative genetic interactions with irc6∆ are enriched for genes related to membrane traffic and nuclear processes, consistent with diverse cellular roles for Irc6.

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AbstractThe review by Kaczmarek et al. describes new functions of the Arf1 small G protein and its activator GBF1, beyond their well‐known role in formation of retrograde transport vesicles between the endoplasmic reticulum and the Golgi apparatus. These new functions include roles in lipid droplet metabolism, clathrin‐independent endocytosis, mitochondrial dynamics and transport of organelles along microtubules.

COPB2 suppresses cell proliferation and induces cell cycle arrest in human colon cancer by regulating cell cycle-related proteins.

Coat proteins (COPs), including the major types clathrin, COPI and COPII, play a considerable role in intracellular transport by initiating the formation of transport vesicles. Coatomer protein complex subunit β2 (COPB2) is one of the seven subunits that make up a COPI complex. In the present study, we found that COPB2 was highly expressed in human colon cancer specimens. However, to date, there have been no reports describing the functions of COPB2 in human colon cancer cells. In this study, we analyzed the functions of COPB2 in the proliferation and cell cycle arrest of human RKO and HCT116 colon cancer cells by using lentivirus-mediated RNAi infection. Our results demonstrated that the silencing of COPB2 in vitro could inhibit the proliferation and colony formation abilities of RKO and HCT116 cells. Furthermore, measurement of cell cycle distribution indicated that the downregulation of COPB2 could induce G0/G1 or S phase cell cycle arrest by regulating cell cycle-related proteins. In conclusion, our results suggest that COPB2 plays a key role in the proliferation and cell cycle progression of human RKO and HCT116 colon cancer cells, thus indicating that COPB2 might be a potential therapeutic target for the treatment of human colon cancer.

The Mobility of AMPA-Type Glutamate Receptors as a Key Factor in the Expression and Maintenance of Synaptic Potentiation

The dynamics of the expression of post-tetanic potentiation of the focal response (the P spike) in the synaptic system consisting of Schaffer collaterals and field CA1 pyramidal neurons were studied in hippocampal slices from ICR mice. Potentiation was induced by high-frequency stimulation of Schaffer collaterals (1 sec, 100 Hz). At 10 sec, all slices in the group demonstrated an increase in the amplitude of the P spike in response to test stimuli. Slices with sharp increases in amplitude to maximal levels were seen, along with slices showing smooth increases in P-spike amplitude. Studies with the transport vesicle formation blocker brefeldin A assessed the relationship between the dependence of the phase of post-tetanic potentiation on intracellular transport. Brefeldin A was added to the perfusion solution before tetanic stimulation. This treatment of slices for 60 min had no effect on the magnitude of the basal P spike or the expression of post-tetanic potentiation, though it impaired the subsequent phase, i.e., maintenance of potentiation. A drop in P-spike amplitude was seen. The integrative activity of the system supporting the delivery of glutamate receptors to dendritic spines in the numbers and at the times required for rapid changes in and prolonged maintenance of the effectiveness of neurotransmission is discussed.

NS1619 regulates the expression of caveolin-1 protein in a time-dependent manner via ROS/PI3K/PKB/FoxO1 signaling pathway in brain tumor microvascular endothelial cells

NS1619, a calcium-activated potassium channel (Kca channel) activator, can selectively and time-dependently accelerate the formation of transport vesicles in both the brain tumor capillary endothelium and tumor cells within 15min of treatment and then increase the permeability of the blood-brain tumor barrier (BTB). However, the mechanism involved is still under investigation. Using a rat brain glioma (C6) model, the expression of caveolin-1, FoxO1 and p-FoxO1 protein were examined at different time points after intracarotid infusion of NS1619 at a dose of 30μg/kg/min. Internalization of Cholera toxin subunit (CTB) labeled fluorescently was monitored by flow cytometry. The expression of caveolin-1 and FoxO1 protein at tumor microvessels was enhanced and caveolae-mediated CTB endocytosis was increased by NS1619 infusion for 15min. Compared with the 15min group, the expression of caveolin-1 protein was significantly decreased and the level of phosphorylation of FoxO1 was significantly increased in the NS1619 2h group. In addition, inhibitors of reactive oxygen species (ROS) or PI3K or PKB significantly attenuated the level of FoxO1 phosphorylation and also increased the expression of caveolin-1 protein in Human Brain Microvascular Endothelial Cells (HBMECs) cocultured with human glioma cells (U87) 2h after NS1619 treatment. This led to the conclusion that NS1619-mediated transport vesicle increase is, at least partly, related to the ROS/PI3K/PKB/FoxO1 signaling pathway.

Membrane bending by protein crowding is affected by protein lateral confinement

Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches.

Durable protein lattices of clathrin that can be functionalized with nanoparticles and active biomolecules.

Biological molecules that self-assemble and interact with other molecules are attractive building blocks for engineering biological devices. DNA has been widely used for the creation of nanomaterials, but the use of proteins remains largely unexplored. Here, we show that clathrin can form homogeneous and extended two-dimensional lattices on a variety of substrates, including glass, metal, carbon and plastic. Clathrin is a three-legged protein complex with unique self-assembling properties and is relevant in the formation of membrane transport vesicles in eukaryotic cells. We used a fragment of the adaptor protein epsin to immobilize clathrin lattices on the substrates. The lattices span multiple square millimetres with a regular periodicity of 30 nm and can be functionalized via modified subunits of clathrin with either inorganic nanoparticles or active enzymes. The lattices can be stored for months after crosslinking and stabilization with uranyl acetate. They could be dehydrated and rehydrated without loss of function, offering potential applications in sensing and as biosynthetic reactors.

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.

Dynamin-related proteins in plant post-Golgi traffic.

Membrane traffic between two organelles begins with the formation of transport vesicles from the donor organelle. Dynamin-related proteins (DRPs), which are large multidomain GTPases, play crucial roles in vesicle formation in post-Golgi traffic. Numerous in vivo and in vitro studies indicate that animal dynamins, which are members of DRP family, assemble into ring- or helix-shaped structures at the neck of a bud site on the donor membrane, where they constrict and sever the neck membrane in a GTP hydrolysis-dependent manner. While much is known about DRP-mediated trafficking in animal cells, little is known about it in plant cells. So far, two structurally distinct subfamilies of plant DRPs (DRP1 and DRP2) have been found to participate in various pathways of post-Golgi traffic. This review summarizes the structural and functional differences between these two DRP subfamilies, focusing on their molecular, cellular and developmental properties. We also discuss the molecular networks underlying the functional machinery centering on these two DRP subfamilies. Furthermore, we hope that this review will provide direction for future studies on the mechanisms of vesicle formation that are not only unique to plants but also common to eukaryotes.

Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation.

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.

ArfGAPs: key regulators for receptor sorting.

Mammalian cells have many membranous organelles that require proper composition of proteins and lipids. Cargo sorting is a process required for transporting specific proteins and lipids to appropriate organelles, and if this process is disrupted, organelle function as well as cell function is disrupted. ArfGAP family proteins have been found to be critical for receptor sorting. In this review, we summarize our recent knowledge about the mechanism of cargo sorting that require function of ArfGAPs in promoting the formation of transport vesicles, and discuss the involvement of specific ArfGAPs for the sorting of a variety of receptors, such as MPR, EGFR, TfR, Glut4, TRAIL-R1/DR4, M5-muscarinic receptor, c-KIT, rhodopsin and β1-integrin. Given the importance of many of these receptors to human disease, the studies of ArfGAPs may provide novel therapeutic strategies in addition to providing mechanistic insight of receptor sorting.