Golgi Compartment Research Articles

STeerING PI4P for innate immune activation

STING transverses the endoplasmic reticulum (ER), Golgi, and endosomal compartments before its degradation within the lysosomes. In this issue of Immunity, Fang etal. demonstrate that the enrichment of cholesterol and sphingomyelin in the trans-Golgi network and endosomes mediated by the ARMH3-PI4KB-PI4P pathway plays a pivotal role in STING activation under cGAS-dependent and -independent conditions.

Heparin and calreticulin interact on the surface of early G0/G1 dividing rat mesangial cells to regulate hyperglycemic glucose-induced responses

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at ∼70kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division.

Mapping localization of 21 endogenous proteins in the Golgi apparatus of rodent neurons

The Golgi apparatus is the major sorting hub in the secretory pathway and particularly important for protein sorting in neurons. Knowledge about protein localization in Golgi compartments is largely based on work in cell lines. Here, we systematically compared protein localization of 21 endogenous proteins in the Golgi apparatus of mouse neurons using confocal microscopy and line scan analysis. We localized these proteins by measuring the distance relative to the canonical TGN marker TGN38. Based on this, proteins fell into three groups: upstream of, overlapping with or downstream of TGN38. Seven proteins showed complete overlap with TGN38, while proteins downstream of TGN38 were located at varying distances from TGN38. Proteins upstream of TGN38 were localized in between TGN38 and the cis-/medial Golgi markers Giantin and GM130. This localization was consistent with protein function. Our data provide an overview of the relative localization of endogenous proteins in the Golgi of primary mouse neurons.

Understanding the pancake-like morphology of Golgi apparatus compartments.

The Golgi Apparatus is a highly conserved membrane-bound organelle involved in a complex cascade of post-translational protein modifications, with the function being closely tied to morphology. Based on bending energy minimization, one would expect membrane compartments of low volume-to-surface ratios, as found in the Golgi compartments (cisterna), to form a cup-like stomatocyte. In nature, however, the Golgi compartments have a flat pancake-like morphology. We investigate additional factors required for this flat pancake-like morphology. Therefore, we adhere giant unilamellar vesicles (GUVs) of varying volume-to-surface ratios to flat surfaces with varying adhesion energies. We find that by combining bending energy with adhesion, pancake-like shapes at lower volume-to-surface ratios can be achieved. However, it does not result in the ultra-flat morphology observed in the Golgi, implying that factors additional to bending and adhesion energy, such as the presence of rim stabilizing proteins or lipids need to be considered.

Inhibition of Hyper-ActivatedLysosomes Restores Youthful Properties and Functions in Old Hematopoietic Stem Cells

Aging is the primary risk factor for many late-onset diseases including myeloid malignancies and immune deficiencies of the elderly. Aging of hematopoietic stem cells (HSCs) is thought to be in part responsible for these blood disorders. As quiescence maintains HSCs' health and function throughout life, loss of quiescence with age is likely implicated in defects of old HSCs including their myeloid-biased lineage commitment. Despite major progress, mechanisms that promote aging of HSCs remain to be elucidated. We have identified dynamic lysosomal modulations as a switch in the precise control of HSC quiescence vs. priming/activation in young HSCs. Here we find that lysosomal properties and functions are compromised in old HSCs. We find that lysosomes are relatively depleted in old HSCs, as evaluated by high resolution confocal microscopy analyses of immunofluorescence (IF) staining of lysosomal-associated membrane protein (LAMP)-1 and the more specific LAMP-2 markers. Notably, lysosomal properties are altered in old HSCs beyond what is known of their function in autophagy. We find increased recruitment of damage-associated proteins Galectin 1 and 2 to lysosomes in old relative to young highly purified LSKCD150+CD48- HSCs indicating increased lysosomal damage in old HSCs. In addition, lysosomal LC3 flux that is an indication of lysosomal activity was increased by two-fold in highly purified old relative to young HSCs. This was in line with old HSCs' alteration of expression of lysosomal enzymes Cytochalasin C and D and the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase). The increased lysosomal activity is supported by greatly acidified lysosomes as well as enhanced lysosomal degradation capacity of cargo in old relative to young HSCs. An 18-hour inhibition of lysosomal acidification by repressing v-ATPase' activity using specific and high affinity v-ATPase inhibitor, ex vivo, led to a dramatic enrichment of old HSCs in large lysosomes, restoration of their properties and reducing their Galectin protein burden similar to what was observed in young quiescent and healthy HSCs. This was specific to lysosomes in the endo-lysosomal pathway as the effect on the Golgi and endoplasmic reticulum compartments in HSCs was relatively negligeable. The v-ATPase inhibitory treatment increased substantially the fraction of non-dividing old HSCs in culture, and the frequency of LTC-ICs recovered in limiting dilution analysis of old HSCs. Importantly, a 4-day ex vivo inhibition of lysosomal activity increased old HSCs' in vivo competitive repopulation ability by over 8-fold over 21 weeks and balanced the production of lineages downstream of old HSCs. Remarkably, only v-ATPase inhibitor- but not vehicle-treated old HSCs sustained survival in lethally irradiated secondarily transplanted mice and maintained self-renewal. v-ATPase inhibition normalized the expression of TFEB, master regulator of lysosomal biogenesis, suppressed CDK6 nuclear expression, normalized metabolic properties of old HSCs, including mTOR activity and glucose uptake, normalized expression of histone marks H4K16ac, H3K27ac and H3K56ac in old HSCs, as well as signs of intrinsic inflammation. Overall, our work indicates that inhibiting lysosomal hyper-activation restores optimum mitochondrial sequestration in old HSCs' lysosomes, leading to reduced inflammation and restitution of stem cell function in old HSCs. These findings may be exploited for improving HSC function in the elderly.

Regulation of myeloid and lymphoid cell development by O-glycans on Notch.

Notch signaling via NOTCH1 stimulated by Delta-like ligand 4 (DLL4) is required for the development of T cells in thymus, and NOTCH2 stimulated by Notch ligand DLL1 is required for the development of marginal zone (MZ) B cells in spleen. Notch signaling also regulates myeloid cell production in bone marrow and is an essential contributor to the generation of early hematopoietic stem cells (HSC). The differentiation program in each of these cellular contexts is optimized by the regulation of Notch signaling strength by O-glycans attached to epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors. There are three major types of O-glycan on NOTCH1 and NOTCH2 - O-fucose, O-glucose and O-GlcNAc. The initiating sugar of each O-glycan is added in the endoplasmic reticulum (ER) by glycosyltransferases POFUT1 (fucose), POGLUT1/2/3 (glucose) or EOGT (GlcNAc), respectively. Additional sugars are added in the Golgi compartment during passage through the secretory pathway to the plasma membrane. Of particular significance for Notch signaling is the addition of GlcNAc to O-fucose on an EGF repeat by the Fringe GlcNAc-transferases LFNG, MFNG or RFNG. Canonical Notch ligands (DLL1, DLL4, JAG1, JAG2) expressed in stromal cells bind to the extracellular domain of Notch receptors expressed in hematopoietic stem cells and myeloid and lymphoid progenitors to activate Notch signaling. Ligand-receptor binding is differentially regulated by the O-glycans on Notch. This review will summarize our understanding of the regulation of Notch signaling in myeloid and lymphoid cell development by specific O-glycans in mice with dysregulated expression of a particular glycosyltransferase and discuss how this may impact immune system development and malignancy in general, and in individuals with a congenital defect in the synthesis of the O-glycans attached to EGF repeats.

On the nature of the plant ER exit sites.

In plants, the endoplasmic reticulum (ER) and Golgi bodies are not only in close proximity, but are also physically linked. This unique organization raises questions about the nature of the transport vectors carrying cargo between the two organelles. Same as in metazoan and yeast cells, it was suggested that cargo is transported from the ER to Golgi cisternae via COPII-coated vesicles produced at ribosome-free ER exit sites (ERES). Recent developments in mammalian cell research suggest, though, that COPII helps to select secretory cargo, but does not coat the carriers leaving the ER. Furthermore, it was shown that mammalian ERES expand into a tubular network containing secretory cargo, but no COPII components. Because of the close association of the ER and Golgi bodies in plant cells, it was previously proposed that ERES and the Golgi comprise a secretory unit that travels over or with a motile ER membrane. In this study, we aimed to explore the nature of ERES in plant cells and took advantage of high-resolution confocal microscopy and imaged ERES labelled with canonical markers (Sar1a, Sec16, Sec24). We found that ERES are dynamically connected to Golgi bodies and most likely represent pre-cis-Golgi cisternae. Furthermore, we showed fine tubular connections from the ER to Golgi compartments (ERGo tubules) as well as fine protrusions from ERES/Golgi cisternae connecting with the ER. We suggest that these tubules observed between the ER and Golgi as well as between the ER and ERES are involved in stabilizing the physical connection between ER and ERES/Golgi cisternae, but may also be involved in cargo transport from the ER to Golgi bodies.

Claudin-4 localization in epithelial ovarian cancer

Claudin-4, a protein with the structure of classic claudins most often found in cell-cell junctions, is frequently overexpressed in epithelial cancers where its localization has not been studied. In this study we aimed to find out where this membrane protein is localized in an ovarian tumor model, OVCAR3 cells, that express high levels of the protein. Immunohistochemical studies showed claudin-4 staining in a perinuclear region, at most plasma membranes and in cytoplasmic puncta. Native claudin-4 did not overlap with phosphorylated claudin-4, which was partially located in focal adhesions. Using claudin-4 BioID technology we confirmed that large amounts of claudin-4 are localized to the Golgi compartment, including in dispersed Golgi in cells where claudin-4 is partially knocked down and in dividing cells. Claudin-4 appears to be present in the vicinity of several types of cell-cell junctions, but there is no evidence that it forms tight junctions in these tumor cells. Both claudin-4, the Golgi marker GM130, and the plasma membrane receptor Notch2 were found in dispersed Golgi in dividing cells. This definition of the cellular architecture of claudin-4 should provide a framework for better understanding of the function of claudin-4 in tumor cells and its molecular interactions.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

LMAN1, a Negative Regulator of the Response to House Dust Mite

Abstract We have utilized an unbiased receptor capture approach (CaptiRec) to screen for receptors on dendritic cells which directly bind HDM. Using this technique, we have identified LMAN1 (Lectin, Mannose Binding 1) as a candidate receptor for HDM. LMAN1 is a mannose binding lectin which is primarily known to act as a cargo transporter between the ER, ERGIC, and Golgi compartments. The role played by LMAN1 in the recognition of and response to HDM, is currently unknown. We verify the ability of LMAN1 to directly bind HDM proteins biochemically and in a cellular context. Furthermore, we present evidence to demonstrate that LMAN1 downregulates NF-κB responses in response to inflammatory cytokines and to HDM. Loss of LMAN1 on DCs used for allergic sensitization leads to exacerbated airway inflammation in a HDM model of allergic asthma. Lastly, peripheral DCs of asthmatics show a significant reduction in expression of LMAN1 compared to healthy controls. These findings have potential implications for the development of novel therapeutic interventions for allergy and allergic asthma. Supported by American Lung Association Innovation Award IA-695632

Deficiency in coatomer complex I causes aberrant activation of STING signalling

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.

TMED2 binding restricts SMO to the ER and Golgi compartments.

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.

Identification of Marek's disease virus pUL56 homologue and analysis of critical amino acid stretches indispensable for its intracellular localization

Marek's disease virus (MDV) is considered a unique member of the Alphaherpesvirinae subfamily that induces rapid onset of T cell lymphoma in chickens. Compared with other conserved UL56 gene homologues of herpesviruses, little is known about the roles of MDV UL56 gene, while recent studies of mammalian herpesvirus pUL56 proteins have revealed their involvement in promoting ubiquitination of the Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) -like E3 ubiquitin ligases for proteasomal degradation and in modulating host immune responses. To determine the expression kinetics of UL56 gene products, chicken embryo fibroblasts were infected with very virulent or attenuated MDV strain and analyzed by quantitative PCR and Western blotting. During the time course of infection, the levels of UL56 mRNA transcripts increased consistently. At the translational level, the pUL56 protein encoded by UL56 gene was expressed in the size of 32 kDa, which emerged as early as 12 h post-infection (hpi) but otherwise began to wane at 72 hpi thereafter. With the treatment of viral DNA synthesis inhibitors, the pUL56 expression was significantly reduced, featuring the dynamics of a late (γ)-gene product. By confocal imaging, pUL56 was found to reside in the Golgi compartment. Both the L-domain motifs and the C-terminal tail-anchored transmembrane were essential for its intracellular localization. Noticeably, pUL56 co-localized with a truncated mutant of the chicken Nedd4-like family protein harboring only the WW domains; however, co-immunoprecipitation assay established no direct interaction between them, and the ectopic expression of pUL56 did not alter the abundance of endogenous Nedd4-like protein. Overall, the present study provides a caveat that the pUL56 homologues of different herpesviruses with structural similarities might vary in expression patterns and probably in functional consequences. For this reason, further investigation should be encouraged to focus on the potential association between UL56 gene and MDV pathogenesis in the context of engineered viral mutants.

Interactions of Severe Acute Respiratory Syndrome Coronavirus 2 Protein E With Cell Junctions and Polarity PSD-95/Dlg/ZO-1-Containing Proteins

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ-binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, and LNX2) and MPP5/PALS1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4, and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in the E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

Linking GOLPH3 and Extracellular Vesicles Content-a Potential New Route in Cancer Physiopathology and a Promising Therapeutic Target is in Sight?

Golgi phosphoprotein 3 (GOLPH3), a highly conserved phosphatidylinositol 4-phosphate effector, is required for maintenance of Golgi architecture, vesicle trafficking, and Golgi glycosylation. GOLPH3 overexpression has been reported in several human solid cancers, including glioblastoma, breast cancer, colorectal cancer, nonsmall cell lung cancer, epithelial ovarian cancer, prostate cancer, gastric cancer, and hepatocellular carcinoma. Although the molecular mechanisms that link GOLPH3 to tumorigenesis require further investigation, it is likely that GOLPH3 may act by controlling the intracellular movement of key oncogenic molecules, between the Golgi compartments and/or between the Golgi and the endoplasmic reticulum. Indeed, numerous evidence indicates that deregulation of intracellular vesicle trafficking contributes to several aspects of cancer phenotypes. However, a direct and clear link between extracellular vesicle movements and GOLPH3 is still missing. In the past years several lines of evidence have implicated GOLPH3 in the regulation of extracellular vesicle content. Specifically, a new role for GOLPH3 has emerged in controlling the internalization of exosomes containing either oncogenic proteins or noncoding RNAs, especially micro-RNA. Although far from being elucidated, growing evidence indicates that GOLPH3 does not increase quantitatively the excretion of exosomes, but rather regulates the exosome content. In particular, recent data support a role for GOLPH3 for loading specific oncogenic molecules into the exosomes, driving both tumor malignancy and metastasis formation. Additionally, the older literature indirectly implicates GOLPH3 in cancerogenesis through its function in controlling hepatitis C virus secretion, which in turn is linked to hepatocellular carcinoma formation. Thus, GOLPH3 might promote tumorigenesis in unexpected ways, involving both direct and indirect routes. If these data are further confirmed, the spectrum of action of GOLPH3 in tumor formation will significantly expand, indicating this protein as a strong candidate for targeted cancer therapy.

A conserved clathrin-coated vesicle component, OsSCYL2, regulates plant innate immunity in rice.

Clathrin‐mediated vesicle trafficking (CMVT) is a fundamental process in all eukaryotic species, and indispensable to organism's growth and development. Recently, it has been suggested that CMVT also plays important roles in the regulation of plant immunity. However, the molecular link between CMVT and plant immunity is largely unknown. SCY1‐LIKE2 (SCYL2) is evolutionally conserved among the eukaryote species. Loss‐of‐function of SCYL2 in Arabidopsis led to severe growth defects. Here, we show that mutation of OsSCYL2 in rice gave rise to a novel phenotype—hypersensitive response‐like (HR) cell death in a light‐dependent manner. Although mutants of OsSCYL2 showed additional defects in the photosynthetic system, they exhibited enhanced resistance to bacterial pathogens. Subcellular localisation showed that OsSCYL2 localized at Golgi, trans‐Golgi network and prevacuolar compartment. OsSCYL2 interacted with OsSPL28, subunit of a clathrin‐associated adaptor protein that is known to regulate HR‐like cell death in rice. We further showed that OsSCYL2–OsSPL28 interaction is mediated by OsCHC1. Collectively, we characterized a novel component of the CMVT pathway in the regulation of plant immunity. Our work also revealed unidentified new functions of the very conserved SCYL2. It thus may provide new breeding targets to achieve both high yield and enhanced resistance in crops.

Palmitoylated small GTPase ARL15 is translocated within Golgi network during adipogenesis.

ABSTRACTThe small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human β-cell line and loss-of-function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction.This article has an associated First Person interview with the first author of the paper.

Golgi membrane protein Erd1 Is essential for recycling a subset of Golgi glycosyltransferases.

Protein glycosylation in the Golgi is a sequential process that requires proper distribution of transmembrane glycosyltransferase enzymes in the appropriate Golgi compartments. Some of the cytosolic machinery required for the steady-state localization of some Golgi enzymes are known but existing models do not explain how many of these enzymes are localized. Here, we uncover the role of an integral membrane protein in yeast, Erd1, as a key facilitator of Golgi glycosyltransferase recycling by directly interacting with both the Golgi enzymes and the cytosolic receptor, Vps74. Loss of Erd1 function results in mislocalization of Golgi enzymes to the vacuole/lysosome. We present evidence that Erd1 forms an integral part of the recycling machinery and ensures productive recycling of several early Golgi enzymes. Our work provides new insights on how the localization of Golgi glycosyltransferases is spatially and temporally regulated, and is finely tuned to the cues of Golgi maturation.

An online gathering about the latest on molecular membrane biology

An online gathering about the latest on molecular membrane biology

Induced pluripotent stem cells for defining Parkinsonian patient subtypes: a further step toward precision medicine.

Induced pluripotent stem cells for defining Parkinsonian patient subtypes: a further step toward precision medicine.