Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion.

The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1ß and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1ß induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.

Biallelic variants in VPS50 cause a neurodevelopmental disorder with neonatal cholestasis.

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes are membrane-tethering heterotetramers located at the trans-Golgi network and recycling endosomes, respectively. GARP and EARP share the three subunits VPS51, VPS52 and VPS53, while VPS50 is unique to EARP and VPS54 to GARP. Retrograde transport of endosomal cargos to the trans-Golgi network is mediated by GARP and endocytic recycling by EARP. Here we report two unrelated individuals with homozygous variants in VPS50, a splice variant (c.1978-1G>T) and an in-frame deletion (p.Thr608del). Both patients had severe developmental delay, postnatal microcephaly, corpus callosum hypoplasia, seizures and irritability, transient neonatal cholestasis and failure to thrive. Light and transmission electron microscopy of liver from one revealed the absence of gamma-glutamyltransferase at bile canaliculi, with mislocalization to basolateral membranes and abnormal tight junctions. Using patient-derived fibroblasts, we identified reduced VPS50 protein accompanied by reduced levels of VPS52 and VPS53. While the transferrin receptor internalization rate was normal in cells of both patients, recycling of the receptor to the plasma membrane was significantly delayed. These data underscore the importance of VPS50 and/or the EARP complex in endocytic recycling and suggest an additional function in establishing cell polarity and trafficking between basolateral and apical membranes in hepatocytes. Individuals with biallelic hypomorphic variants in VPS50, VPS51 or VPS53 show an overarching neurodegenerative disorder with severe developmental delay, intellectual disability, microcephaly, early-onset epilepsy and variable atrophy of the cerebellum, cerebrum and/or brainstem. The term 'GARP/EARP deficiency' designates disorders in such individuals.

In pancreatic ß-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi.

Pancreatic ß-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic ß-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.

Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network.

Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/trans-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.

An integrative view on vacuolar pH homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation.

The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar-type H+ -translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans-Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca2+ /H+ -antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP-dependent Ca2+ -pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work.

LRRK2 and the Endolysosomal System in Parkinson's Disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson's disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

The mysterious life of the plant trans-Golgi network: advances and tools to understand it better.

By being at the interface of the exocytic and endocytic pathways, the plant trans-Golgi network (TGN) is a multitasking and highly diversified organelle. Despite governing vital cellular processes, the TGN remains one of the most uncharacterized organelle of plant cells. In this review, we highlight recent studies that have contributed new insights and to the generation of markers needed to answer several important questions on the plant TGN. Several drugs specifically affecting proteins critical for the TGN functions have been extremely useful for the identification of mutants of the TGN in the pursuit to understand how the morphology and the function of this organelle are controlled. In addition to these chemical tools, we review emerging microscopy techniques that help visualize the TGN at an unpreceded resolution and appreciate the heterogeneity and dynamics of this organelle in plant cells.

Differential sorting behavior for soluble and transmembrane cargoes at the trans-Golgi network in endocrine cells.

Regulated secretion of neuropeptides and peptide hormones by secretory granules (SGs) is central to physiology. Formation of SGs occurs at the trans-Golgi network (TGN) where their soluble cargo aggregates to form a dense core, but the mechanisms controlling the sorting of regulated secretory cargoes (soluble and transmembrane) away from constitutively secreted proteins remain unclear. Optimizing the use of the retention using selective hooks method in (neuro-)endocrine cells, we now quantify TGN budding kinetics of constitutive and regulated secretory cargoes. We further show that, by monitoring two cargoes simultaneously, it becomes possible to visualize sorting to the constitutive and regulated secretory pathways in real time. Further analysis of the localization of SG cargoes immediately after budding from the TGN revealed that, surprisingly, the bulk of two studied transmembrane SG cargoes (phogrin and VMAT2) does not sort directly onto SGs during budding, but rather exit the TGN into nonregulated vesicles to get incorporated to SGs at a later step. This differential behavior of soluble and transmembrane cargoes suggests a more complex model of SG biogenesis than anticipated.

Distinct anterograde trafficking pathways of BACE1 and amyloid precursor protein from the TGN and the regulation of amyloid-ß production.

Processing of amyloid precursor protein (APP) by the ß-secretase BACE1 is the initial step of the amyloidogenic pathway to generate amyloid-ß (Aß). Although newly synthesized BACE1 and APP are transported along the secretory pathway, it is not known whether BACE1 and APP share the same post-Golgi trafficking pathways or are partitioned into different transport routes. Here we demonstrate that BACE1 exits the Golgi in HeLa cells and primary neurons by a pathway distinct from the trafficking pathway for APP. By using the Retention Using Selective Hooks system, we show that BACE1 is transported from the trans-Golgi network to the plasma membrane in an AP-1- and Arf1/4-dependent manner. Subsequently, BACE1 is endocytosed to early and recycling endosomes. Perturbation of BACE1 post-Golgi trafficking results in an increase in BACE1 cleavage of APP and increased production of both Aß40 and Aß42. These findings reveal that Golgi exit of BACE1 and APP in primary neurons is tightly regulated, resulting in their segregation along different transport routes, which limits APP processing.

Syntaxin 16's Newly Deciphered Roles in Autophagy.

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.

A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells.

BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function.

The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in attrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

Overexpression of trans-Golgi network t-SNAREs rescues vacuolar trafficking and TGN morphology defects in a putative tethering factor mutant.

The trans-Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN-localized coiled-coil protein TNO1 is a putative tethering factor that interacts with the TGN t-SNARE SYP41 and is required for correct localization of the SYP61 t-SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41-SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41-SYP61 t-SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.

Rho-of-plant activated root hair formation requires Arabidopsis YIP4a/b gene function.

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.

Action of Arl1 GTPase and golgin Imh1 in Ypt6-independent retrograde transport from endosomes to the trans-Golgi network.

The Arf and Rab/Ypt GTPases coordinately regulate membrane traffic and organelle structure by regulating vesicle formation and fusion. Ample evidence has indicated that proteins in these two families may function in parallel or complementarily; however, the manner in which Arf and Rab/Ypt proteins perform interchangeable functions remains unclear. In this study, we report that a Golgi-localized Arf, Arl1, could suppress Ypt6 dysfunction via its effector golgin, Imh1, but not via the lipid flippase Drs2. Ypt6 is critical for the retrograde transport of vesicles from endosomes to the trans-Golgi network (TGN), and its mutation leads to severe protein mislocalization and growth defects. We first overexpress the components of the Arl3-Syt1-Arl1-Imh1 cascade and show that only Arl1 and Imh1 can restore endosome-to-TGN trafficking in ypt6-deleted cells. Interestingly, increased abundance of Arl1 or Imh1 restores localization of the tethering factor Golgi associated retrograde-protein (GARP) complex to the TGN in the absence of Ypt6. We further show that the N-terminal domain of Imh1 is critical for restoring GARP localization and endosome-to-TGN transport in ypt6-deleted cells. Together, our results reveal the mechanism by which Arl1-Imh1 facilitates the recruitment of GARP to the TGN and compensates for the endosome-to-TGN trafficking defects in dysfunctional Ypt6 conditions.

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity.

Glycosphingolipid (GSL) metabolism is involved in various physiological processes, including all major cell signaling pathways, and its dysregulation is linked to some diseases. The four-phosphate adaptor protein FAPP2-mediated glucosylceramide (GlcCer) transport for complex GSL synthesis has been studied extensively. However, the molecular machinery of FAPP2 as a GlcCer-transferring protein remains poorly defined. Here, we identify a Golgi-resident protein, acyl-coenzyme A binding domain containing 3 (ACBD3), as an interacting partner of FAPP2. We find that ACBD3 knockdown leads to dramatic Golgi fragmentation, which subsequently causes FAPP2 dispersal throughout the cytoplasm and a decreased localization at trans-Golgi network. The further quantitative lipidomic analysis indicates that ACBD3 knockdown triggers abnormal sphingolipid metabolism. Interestingly, the expression of siRNA-resistant full-length ACBD3 can rescue these defects caused by ACBD3 knockdown. These data reveal critical roles for ACBD3 in maintaining the integrity of Golgi morphology and cellular sphingolipid homeostasis and establish the importance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis.

Endosomal sorting and trafficking, the retromer complex and neurodegeneration.

The retromer is a highly conserved multimeric protein complex present in all eukaryotic cells whose activity is essential for regulating the recycling and retrieval of numerous protein cargos from the endosome to trans-Golgi network or the cell surface. In recent years, molecular and genomic studies have provided evidence that aberrant regulation of endosomal protein sorting and trafficking secondary to a dysfunction of the retromer complex could be implicated in the pathogenesis of several neurodegenerative diseases. Thus, deficiency or mutations in one or more protein components of the retromer leads to increased accumulation of protein aggregates, as well as enhanced cellular neurotoxicity. In this review, we will discuss the structure and function of the retromer complex and its neurobiology, its relevance to key molecules involved in neurodegeneration and the potential role that it plays in the development of two major neurodegenerative disorders, Parkinson's disease and Alzheimer's disease. Finally, we will discuss the viability of targeting the retromer via pharmacological chaperones or genetic approaches to enhance or restore its function as a novel and unifying disease-modifying strategy against these diseases.

Plant TGN in the stress response: a compartmentalized overview.

The cellular responses to abiotic and biotic stress rely on the regulation of vesicle trafficking to ensure the correct localization of proteins specialized in sensing stress stimuli and effecting the response. Several studies have implicated the plant trans-Golgi network (TGN)-mediated trafficking in different types of biotic and abiotic stress responses; however, the underlying molecular mechanisms are poorly understood. Further, the identity, specialization and stress-relevant cargo transported by the TGN subcompartments involved in stress responses await more in depth characterization. This review presents TGN trafficking players implicated in stress and discusses potential avenues to understand the role of this dynamic network under such extreme circumstances.

Phototracking Vaccinia Virus Transport Reveals Dynamics of Cytoplasmic Dispersal and a Requirement for A36R and F12L for Exit from the Site of Wrapping.

The microtubule cytoskeleton is a primary organizer of viral infections for delivering virus particles to their sites of replication, establishing and maintaining subcellular compartments where distinct steps of viral morphogenesis take place, and ultimately dispersing viral progeny. One of the best characterized examples of virus motility is the anterograde transport of the wrapped virus form of vaccinia virus (VACV) from the trans-Golgi network (TGN) to the cell periphery by kinesin-1. Yet many aspects of this transport event are elusive due to the speed of motility and the challenges of imaging this stage at high resolution over extended time periods. We have established a novel imaging technology to track virus transport that uses photoconvertible fluorescent recombinant viruses to track subsets of virus particles from their site of origin and determine their destination. Here we image virus exit from the TGN and their rate of egress to the cell periphery. We demonstrate a role for kinesin-1 engagement in regulating virus exit from the TGN by removing A36 and F12 function, critical viral mediators of kinesin-1 recruitment to virus particles. Phototracking viral particles and components during infection is a powerful new imaging approach to elucidate mechanisms of virus replication.