ER Morphology Research Articles

Ankle2, a Target of Zika Virus, Controls Asymmetric Cell Division of Neuroblasts and Uncovers a Novel Microcephaly Pathway

Neuroblasts in flies divide asymmetrically by establishing polarity, distributing cell fate determinants asymmetrically, and positioning their spindle for cell division. The apical complex contains aPKC, Bazooka (Par3), and Par6, and its activity depends on L(2)gl. We show that Ankle2 interacts with L(2)gl and affects aPKC. Reducing Ankle2 levels disrupts ER and nuclear envelope morphology, releasing the kinase Ballchen/VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen/VRK1 or l(2)gl suppresses the loss of Ankle2 and restores viability and brain size. The Zika virus NS4A protein interacts with Drosophila Ankle2 and VRK1 in dividing neuroblasts. Human mutational studies implicate this neural cell division pathway in microcephaly and motor neuron disease. In summary, NS4A, ANKLE2, VRK1 and LLGL1 define a novel pathway that impinges on asymmetric determinants of neural stem cell division.

Porcine circovirus type 2 ORF5 protein induces endoplasmic reticulum stress and unfolded protein response in porcine alveolar macrophages.

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.

Emission, Er ion defects and structure of ZnO nanocrystal films prepared by ultrasonic spray pyrolysis

This work focuses on the investigation of near band edge (NBE) and Er ion related emissions, morphology and structure of Er-doped ZnO nanocrystal (NC) films prepared by ultrasonic spray pyrolysis with the different Er contents. The scanning electronic microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and photoluminescence (PL) methods have been used. It is revealed that the crystal structure of ZnO:Er films can be improved at low Er-doping (≤ 2 at%), when Er ions occupy the Zn vacancies with the formation of the substitutional ErZn defects. Simultaneously, the PL intensity of NBE emission enlarges and the PL intensity of green PL band, connected with native deep defects, decreases. Meanwhile, the IR emission via the 4f intra-shell optical transitions (4I13/2–4I15/2) in Er ions at this doping is insignificant.The PL intensities of NBE emission, all visible and IR emission bands, connected with the 4f inner-shell optical transitions in Er ions, have enlarged essentially at higher Er-doping (<5 at%). It is revealed at XPS study that the process of Er ion oxidizing is realized at this Er content. The Er concentration of <5 at% can be considered as an optimal for the bright Er ion emission owing to oxidizing and the formation of a low symmetry oxygen environment around the Er ions. Further Er content enlarging provokes PL intensity decreasing and ZnO crystallinity falling down together with the native defect generation in ZnO NCs. It is expected that deep understanding the Er-doping process will be useful for the strategy development to obtaining ZnO:Er NC films with tailored parameters.

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis.

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. Here, we show that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. We propose that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis.

Inhibition of protein tyrosine phosphatase 1B protects against sevoflurane-induced neurotoxicity mediated by ER stress in developing brain

BackgroundER stress is involved in sevoflurane-induced neurotoxicity. Protein tyrosine phosphatase 1B (PTP1B) resided in the ER membrane is known to regulate ER stress. However, the role of PTP1B in sevoflurane-induced neurotoxicity is unknown. MethodSeven-day-old mice treated with 2.3% sevoflurane for 6 h as animal model. The hippocampal tissues were harvested following sevoflurane exposure for evaluation of ER stress markers with Western blot, ER morphology with transmission electron microscopy and density of dendrite spine with Golgi staining. In another subset of mice, neurocognitive function was assessed 4 weeks after anesthesia using Morris water maze test. We also examined the effects of PTP1B or PERK inhibitor on sevoflurane-induced neurodegeneration in the hippocampus. ResultThe results showed inhibition of PTP1B significantly ameliorated sevoflurane-induced (1) ultrastructural ER alternations and ER stress activation as indicated by upregulation of PERK and eIF2α phosphorylation; (2) increase in cleaved caspase-3 expression; (3) elevated expressions of proinflammatory NF-κB and TNFα;(4) decreases in expression of synaptophysin, Arc and BDNF-TrkB proteins as well as loss of dendrite spine in the hippocampus; and (5) impairment in neurobehavioural performance at adolescence. Similarly, ER stress inhibitor is also found to downregulate eIF2α phosphorylation and neuroinflammation, and increase expressions of synaptic proteins and BDNF-TrkB signaling proteins. ConclusionsOur study shows PTP1B inhibition mitigates sevoflurane-induced neurodegeneration maybe mediated by ER stress in developing brain, and eventually improves cognitive function. This suggests PTP1B inhibition could represent a promising strategy to prevent sevoflurane-induced neurotoxicity.

A putative NEM1 homologue regulates lipid droplet biogenesis via PAH1 in Tetrahymena thermophila

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.

Fat storage-inducing transmembrane (FIT or FITM) proteins are related to lipid phosphatase/phosphotransferase enzymes.

Fat storage-inducing transmembrane (FIT or FITM) proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. Saccharomyces cerevisiae has two FITM homologues: Scs3p and Yft2p. Scs3p was initially identified because deletion leads to inositol auxotrophy, with an unusual sensitivity to addition of choline. This strongly suggests a role for Scs3p in phospholipid biosynthesis. Looking at the FITM family as widely as possible, we found that FITMs are widespread throughout eukaryotes, indicating presence in the last eukaryotic common ancestor. Protein alignments also showed that FITM sequences contain the active site of lipid phosphatase/phosphotransferase (LPT) enzymes. This large family transfers phosphate-containing headgroups either between lipids or in exchange for water. We confirmed the prediction that FITMs are related to LPTs by showing that single amino-acid substitutions in the presumptive catalytic site prevented their ability to rescue growth of the mutants on low inositol/high choline media when over-expressed. The substitutions also prevented rescue of other phenotypes associated with loss of FITM in yeast, including mistargeting of Opi1p, defective ER morphology, and aberrant lipid droplet budding. These results suggest that Scs3p, Yft2p and FITMs in general are LPT enzymes involved in an as yet unknown critical step in phospholipid metabolism.

A novel fluorescent reporter detects plastic remodeling of mitochondria-ER contact sites.

Mitochondria-ER contact sites (MERCs) enable communication between the ER and mitochondria and serve as platforms for many cellular events, including autophagy. Nonetheless, the molecular organization of MERCs is not known, and there is no bona fide marker of these contact sites in mammalian cells. In this study, we designed a genetically encoded reporter using split GFP protein for labeling MERCs. We subsequently analyzed its distribution and dynamics during the cell cycle and under stressful cellular conditions such as starvation, apoptosis and ER stress. We found that MERCs are dynamic structures that undergo remodeling within minutes. Mitochondrial morphology, but not ER morphology, affected the distribution of MERCs. We also found that carbonyl cyanidem-chlorophenyl hydrazone (CCCP) and oligomycin A treatment enhanced MERC formation. The stimulations that led to apoptosis or autophagy increased the MERC signal. By contrast, increasing cellular lipid droplet load did not change the pattern of MERCs.

An Evolutionarily Conserved Phosphatidate Phosphatase Maintains Lipid Droplet Number and ER Morphology But Not Nuclear Morphology

Phosphatidic acid phosphatases are involved in the biosynthesis of phospholipids and triacylglycerol, and also act as transcriptional regulators. Studies to ascertain its role in lipid metabolism and membrane biogenesis are restricted only to Opisthokonta and Archaeplastida. Herein, we report the role of phosphatidate phosphatase (PAH) in Tetrahymena thermophila, belonging to the distantly related Alveolata clade. We identified two PAH homologs in Tetrahymena, TtPAH1, and TtPAH2. TtPAH1 maintains lipid droplet number and ER morphology but does not regulate nuclear morphology in Tetrahymena. TtPAH1 rescued all the known defects in yeast PAH1 strain and is conserved functionally between Tetrahymena and yeast. The homologous gene derived from Trypanosoma also rescued the defects of yeast PAH1 strain. Our results indicate that PAH1, previously known to be conserved between Opisthokonts, is also present in a set of distant lineages. Thus, a phosphatase cascade is evolutionarily conserved and is functionally interchangeable across eukaryotic lineages.

Small phosphatidate phosphatase (TtPAH2) of Tetrahymena complements respiratory function and not membrane biogenesis function of yeast PAH1.

Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report the presence of a low-molecular-weight PAH homolog in the single-celled ciliate Tetrahymena thermophila. In vitro phosphatase assay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss of function of TtPAH2 did not affect the growth of Tetrahymena. Unlike other known PAH homologs, TtPAH2 did not regulate lipid droplet number and ER morphology. TtPAH2 did not rescue growth and ER/nuclear membrane defects of the pah1Δ yeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellular functions. Surprisingly, TtPAH2 complemented the respiratory defect in the pah1Δ yeast cells indicating a specific role of TtPAH2 in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family in respiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologs are critical for lipid homeostasis and membrane biogenesis.

An evolutionarily conserved phosphatidate phosphatase maintains lipid droplet number and endoplasmic reticulum morphology but not nuclear morphology.

ABSTRACTPhosphatidic acid phosphatases are involved in the biosynthesis of phospholipids and triacylglycerol, and also act as transcriptional regulators. Studies to ascertain their role in lipid metabolism and membrane biogenesis are restricted to Opisthokonta and Archaeplastida. Here, we report the role of phosphatidate phosphatase (PAH) in Tetrahymena thermophila, belonging to the Alveolata clade. We identified two PAH homologs in Tetrahymena, TtPAH1 and TtPAH2. Loss of function of TtPAH1 results in reduced lipid droplet number and an increase in endoplasmic reticulum (ER) content. It also results in more ER sheet structure as compared to wild-type Tetrahymena. Surprisingly, we did not observe a visible defect in the nuclear morphology of the ΔTtpah1 mutant. TtPAH1 rescued all known defects in the yeast pah1Δ strain and is conserved functionally between Tetrahymena and yeast. The homologous gene derived from Trypanosoma also rescued the defects of the yeast pah1Δ strain. Our results indicate that PAH, previously known to be conserved among Opisthokonts, is also present in a set of distant lineages. Thus, a phosphatase cascade is evolutionarily conserved and is functionally interchangeable across eukaryotic lineages.

Tang-Luo-Ning, a Traditional Chinese Medicine, Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis of Schwann Cells under High Glucose Environment.

Tang-Luo-Ning (TLN) has a definite effect in the clinical treatment of diabetic peripheral neuropathy (DPN). Schwann cells (SCs) apoptosis induced by endoplasmic reticulum stress (ER stress) is one of the main pathogeneses of DPN. This study investigates whether TLN can inhibit SCs apoptosis by inhibiting ER stress-induced apoptosis. Our previous researches have demonstrated that TLN could increase the expression of ER stress marker protein GRP78 and inhibited the expression of apoptosis marker protein CHOP in ER stress. In this study, the results showed that TLN attenuated apoptosis by decreasing Ca2+ level in SCs and maintaining ER morphology. TLN could decrease downstream proteins of CHOP including GADD34 and Ero1α, while it increased P-eIF2α and decreased the upstream proteins of CHOP including P-IRE1α/IRE1α and XBP-1, thereby reducing ER stress-induced apoptosis.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis

Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

Cell biology and the "real world".

Cell biology and the "real world".

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster.

Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.

SNAREs share atlastin’s burden

![Figure][1] Compared with wild-type cells (left), ER morphology is disrupted in yeast lacking both Sey1p and Sec22p (right). SNARE proteins help the dynamin-like GTPase atlastin to fuse ER membranes together, [Lee et al.][2] reveal. ER tubules fuse with each other to form an

Synaptotagmin SYTA Forms ER-Plasma Membrane Junctions that Are Recruited to Plasmodesmata for Plant Virus Movement

Metazoan synaptotagmins are Ca(2+) sensors that regulate exocytosis and endocytosis in various cell types, notably in nerve and neuroendocrine cells [1,2]. Recently, the structurally related extended synaptotagmins were shown to tether the cortical ER to the plasma membrane in human and yeast cells to maintain ER morphology and stabilize ER-plasma membrane (ER-PM) contact sites for intracellular lipid and Ca(2+) signaling [3, 4]. The Arabidopsis synaptotagmin SYTA regulates endocytosis and the ability of plant virus movement proteins (MPs) to alter plasmodesmata to promote virus cell-to-cell transport [5,6]. Yet how MPs modify plasmodesmata, the cellular functions of SYTA and how these aid MP activity, and the proteins essential to form plant cell ER-PM contact sites remain unknown. We addressed these questions using an Arabidopsis SYTA knockdown line syta-1 and a Tobamovirus movement protein MP(TVCV) [5, 7]. We report here that SYTA localized to ER-PM contact sites. These sites were depleted and the ER network collapsed in syta-1, and both reformed upon rescue with SYTA. MP(TVCV) accumulation in plasmodesmata, but not secretory trafficking, was also inhibited in syta-1. During infection, MP(TVCV) recruited SYTA to plasmodesmata, and SYTA and the cortical ER were subsequently remodeled to form viral replication sites adjacent to plasmodesmata in which MP(TVCV) and SYTA directly interacted caged within ER membrane. SYTA also accumulated in plasmodesmata active in MP(TVCV) transport. Our findings show that SYTA is essential to form ER-PM contact sites and suggest that MPs interact with SYTA to recruit these sites to alter plasmodesmata for virus cell-to-cell movement.

An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids.

African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate ‘invariant glycoproteins’ (IGPs). IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism.

Phospholipid biosynthesis increases in RHD3-defective mutants

RHD3, a member of the ER-shaping dynamin-like GTPases, is required in the transition from a cisternal to a tubular ER architecture during cell growth. The aberrant ER morphology in rhd3 mutants may be correlated with alterations of the ER lipid bilayer. We analyzed the lipid fraction of rhd3 mutants at qualitative and quantitative levels. We observed an increase of the amount of phospholipids but also of proteins in the mutants, indicating an overall increase of ER membranes. This increase may indicate that phospholipid biosynthesis is deregulated in rhd3 mutants. It was shown that overexpression of PIS1 and PIS2 (involved in phosphatidylinositol biosynthesis) induces the synthesis of phosphatidylinositol (PI) but also of phosphatidic acid and that overexpression of PIS1 also induces the synthesis of phosphatidylethanolamine and diacylglycerol.1 We wondered whether PIS1 or PIS2 could be linked to the increase of the amount of phospholipids in rhd3 mutants. To answer, we measured the phospholipid composition in the double mutants rhd3–7/pis1 and rhd3–7/pis2. The phospholipid increase in the rhd3 mutant was compensated in rhd3–7/pis1 but not rhd3–7/pis2. Our results suggest a possible deregulation of PIS1 in the rhd3 mutant.