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N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization., Competing Interests: Declaration of competing interests The authors declare no competing or financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone with two isoforms, Hsp90α and Hsp90β. Hsp90β deficiency causes embryonic lethality, whereas Hsp90α deficiency causes few abnormities except male sterility. In this paper, we reported that Hsp90α was exclusively expressed in the retina, testis, and brain. Its deficiency caused retinitis pigmentosa (RP), a disease leading to blindness. In Hsp90α-deficient mice, the retina was deteriorated and the outer segment of photoreceptor was deformed. Immunofluorescence staining and electron microscopic analysis revealed disintegrated Golgi and aberrant intersegmental vesicle transportation in Hsp90α-deficient photoreceptors. Proteomic analysis identified microtubule-associated protein 1B (MAP1B) as an Hsp90α-associated protein in photoreceptors. Hspα deficiency increased degradation of MAP1B by inducing its ubiquitination, causing α-tubulin deacetylation and microtubule destabilization. Furthermore, the treatment of wild-type mice with 17-DMAG, an Hsp90 inhibitor of geldanamycin derivative, induced the same retinal degeneration as Hsp90α deficiency. Taken together, the microtubule destabilization could be the underlying reason for Hsp90α deficiency-induced RP., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K + channels, but only APOL3 exhibits Ca 2+ -dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice ( Oryza sativa ) glutelin precursor accumulation5 ( gpa5 ) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis ( Arabidopsis thaliana ) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N -ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm., (© 2020 American Society of Plant Biologists. All rights reserved.)

Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 μm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.

Lethal small molecules are useful probes to discover and characterize novel cell death pathways and biochemical mechanisms. Here we report that the synthetic oxime-containing small molecule caspase-independent lethal 56 (CIL56) induces an unconventional form of nonapoptotic cell death distinct from necroptosis, ferroptosis, and other pathways. CIL56-induced cell death requires a catalytically active protein S-acyltransferase complex comprising the enzyme ZDHHC5 and an accessory subunit GOLGA7. The ZDHHC5-GOLGA7 complex is mutually stabilizing and localizes to the plasma membrane. CIL56 inhibits anterograde protein transport from the Golgi apparatus, which may be lethal in the context of ongoing ZDHHC5-GOLGA7 complex-dependent retrograde protein trafficking from the plasma membrane to internal sites. Other oxime-containing small molecules, structurally distinct from CIL56, may trigger cell death through the same pathway. These results define an unconventional form of nonapoptotic cell death regulated by protein S-acylation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/ cis -Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition., Competing Interests: The authors declare no competing interest.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth's oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolith-associated polysaccharides ('CAPs') providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate - where the crystals nucleate - are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca 2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca 2+ cations to the mineralization sites., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack., (© 2019 American Society of Plant Biologists. All rights reserved.)

The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn't lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium-maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD-model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium-maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium-maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD-model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS., (© 2019 Wiley Periodicals, Inc.)

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age-related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi-mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro-inflammatory cytokines and extracellular matrix-degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi-related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non-senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans -Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles. IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes., (Copyright © 2019 Melia et al.)

This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 μm or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry., (Copyright © 2019 Sou et al.)

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice ( Oryza sativa ) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13 C- 13 C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops., (© 2019 American Society of Plant Biologists. All rights reserved.)

The plant endoplasmic reticulum-Golgi apparatus is the site of synthesis, assembly, and trafficking of all noncellulosic polysaccharides, proteoglycans, and proteins destined for the cell wall. As grass species make cell walls distinct from those of dicots and noncommelinid monocots, it has been assumed that the differences in cell-wall composition stem from differences in biosynthetic capacities of their respective Golgi. However, immunosorbence-based screens and carbohydrate linkage analysis of polysaccharides in Golgi membranes, enriched by flotation centrifugation from etiolated coleoptiles of maize ( Zea mays ) and leaves of Arabidopsis ( Arabidopsis thaliana ), showed that arabinogalactan-proteins and arabinans represent substantial portions of the Golgi-resident polysaccharides not typically found in high abundance in cell walls of either species. Further, hemicelluloses accumulated in Golgi at levels that contrasted with those found in their respective cell walls, with xyloglucans enriched in maize Golgi, and xylans enriched in Arabidopsis. Consistent with this finding, maize Golgi membranes isolated by flotation centrifugation and enriched further by free-flow electrophoresis, yielded >200 proteins known to function in the biosynthesis and metabolism of cell-wall polysaccharides common to all angiosperms, and not just those specific to cell-wall type. We propose that the distinctive compositions of grass primary cell walls compared with other angiosperms result from differential gating or metabolism of secreted polysaccharides post-Golgi by an as-yet unknown mechanism, and not necessarily by differential expression of genes encoding specific synthase complexes., (© 2019 American Society of Plant Biologists. All rights reserved.)

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-βH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity., (© 2019 Venditti et al.)

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.

Cell motility is critical to biological processes from wound healing to cancer metastasis to embryonic development. The involvement of organelles in cell motility is well established, but the role of organelle positional reorganization in cell motility remains poorly understood. Here we present an automated image analysis technique for tracking the shape and motion of Golgi bodies and cell nuclei. We quantify the relationship between nuclear orientation and the orientation of the Golgi body relative to the nucleus before, during, and after exposure of mouse fibroblasts to a controlled change in cell substrate topography, from flat to wrinkles, designed to trigger polarized motility. We find that the cells alter their mean nuclei orientation, in terms of the nuclear major axis, to increasingly align with the wrinkle direction once the wrinkles form on the substrate surface. This change in alignment occurs within 8 hours of completion of the topographical transition. In contrast, the position of the Golgi body relative to the nucleus remains aligned with the pre-programmed wrinkle direction, regardless of whether it has been fully established. These findings indicate that intracellular positioning of the Golgi body precedes nuclear reorientation during mouse fibroblast directed migration on patterned substrates. We further show that both processes are Rho-associated kinase (ROCK) mediated as they are abolished by pharmacologic ROCK inhibition whereas mouse fibroblast motility is unaffected. The automated image analysis technique introduced could be broadly employed in the study of polarization and other cellular processes in diverse cell types and micro-environments. In addition, having found that the nuclei Golgi vector may be a more sensitive indicator of substrate features than the nuclei orientation, we anticipate the nuclei Golgi vector to be a useful metric for researchers studying the dynamics of cell polarity in response to different micro-environments., Competing Interests: The authors have declared that no competing interests exist.

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedV Sed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypB Sec7 and HypA Trs120 , implying that COPI budding predominates at the SedV Sed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sod VI C1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Condylar cartilage is a joint cartilage essential for smooth jaw movement. The importance of ciliary proteins in condylar cartilage development has been reported. However, little is known about how ciliary proteins control the homeostasis of condylar cartilage. Here we show that intraflagellar transport 20 (IFT20), a ciliary protein, is required for the maintenance of cartilaginous matrix in condylar cartilage. Utilizing NG2-CreER mice expressed in condylar cartilage, we deleted Ift20 by tamoxifen treatment at juvenile-to-adult stages. In wild-type condylar cartilage, IFT20 was robustly produced in the cis-Golgi, but deletion of Ift20 by tamoxifen induction of NG2-CreER (Ift20:NG2-CreER) resulted in reduced cell proliferation and decreased Golgi size in condylar cartilage. Importantly, while the primary cilia were present in cartilage cells in the condylar layers of wild-type mice, no primary cilia were present in the Ift20:NG2-CreER condylar layers. Consistent with this finding, ciliary-mediated Hedgehog signaling was severely attenuated in Ift20 mutant chondrocytes, and thus the production levels of type X collagen were significantly reduced in Ift20:NG2-CreER mice. These results suggest that IFT20 is required for Golgi size and Hedgehog signaling to maintain cartilaginous matrix in condylar cartilage. Our study highlights the unique function of IFT20 in the homeostasis of condylar cartilage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a key role in biosynthesis of vitamin K 2 and coenzyme Q10 using geranylgeranyl diphosphate (GGPP). However, the mechanism by which UBIAD1 participates in tumorigenesis remains unknown. This study show that UBIAD1 interacts with H-Ras, retains H-Ras in the Golgi apparatus, prevents H-Ras trafficking from the Golgi apparatus to the plasma membrane, blocks the aberrant activation of Ras/MAPK signaling, and inhibits the proliferation of bladder cancer cells. In addition, GGPP was required to maintain the function of UBIAD1 in regulating the Ras/ERK signaling pathway. A Drosophila model was employed to confirm the function of UBIAD1/HEIX in vivo. The activation of Ras/ERK signaling at the plasma membrane induced melanotic masses in Drosophila larvae. Our study suggests that UBIAD1 serves as a tumor suppressor in cancer and tentatively reveals the underlying mechanism of melanotic mass formation in Drosophila.

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection 1 . The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis 2 , a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.

Ultrastructural changes on the apical surface of the luminal epithelium of the uterus are known as pinopodes. Their morphology in species and in special species is associated with different results about size, duration, and percentage of surface area covered by pinopodes. The content of pinopodes is different in rodents and humans. In mice and rats pinopodes have many vacuoles and no organelle that extends to the actin stalk above the microvilli. Human pinopodes do not have a large vacuole and contain the golgi complex, a rough endoplasmic reticulum, secretory vesicles, and mitochondria that extend from the entire cell surface. It has been suggested that pinopodes are good markers of endometrial receptivity and implantation window. There are several molecular markers related to the presence of pinopodes, including integrins, leukemia inhibiting factor (LIF), l-selectin, HOXA10, glutaredoxin, glycodelinA, heparin-binding epidermal growth factor, mucins, and microRNAs (miRNAs). Multiple lines of evidence have indicated that miRNAs could affect the expression of LIF and pinopodes in the endometrium and these molecules play key roles in implantation window processes. Here, we have summarized the morphology and function of pinopodes. Moreover, we have highlighted several molecules in relation to pinopodes that could be used as biomarkers., (© 2018 Wiley Periodicals, Inc.)

It is unclear how the two principal functions of the Golgi complex, processing and transport, are spatially organized. Studying such spatial organization by optical imaging is challenging, partially due to the dense packing of stochastically oriented Golgi stacks. Using super-resolution microscopy and markers such as Giantin, we developed a method to identify en face and side views of individual nocodazole-induced Golgi mini-stacks. Our imaging uncovered that Golgi enzymes preferentially localize to the cisternal interior, appearing as a central disk or inner-ring, whereas components of the trafficking machinery reside at the periphery of the stack, including the cisternal rim. Interestingly, conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi. In contrast, bulky cargos were found only at the rim. Our study therefore directly demonstrates the spatial separation of processing and transport functions within the Golgi complex., Competing Interests: HT, AL, SS, LL No competing interests declared, (© 2018, Tie et al.)

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here, we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via sequential elongation and expansion processes. This starts with a synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial-cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

In vitro biocompatibility assessments that consider physiologically appropriate conditions of cell exposure to peritoneal dialysis fluids (PDFs) are still awaited. In this study, we found that fragmentation of Golgi apparatus occurred in a pH-dependent manner within 30-min exposure to five distinct commercially available PDFs, which showed no marked difference in their effects on cell viability in the conventional MTT assay. Fluorescence microscopy analysis of labeling antibody against cis-Golgi protein GM130 indicated that the stacked cisternal structure was maintained in the perinuclear area of both M199 culture medium and a neutral-pH PDF groups. However, this specific structure became partially disassembled over time even in a neutral-pH PDF, and fragmentation was markedly enhanced in cells exposed to neutralized-pH PDFs in correspondence with their intracellular pH; moreover, in acidic PDFs, Golgi staining was diffuse and scattered in the entire cytoplasm and showed partial aggregation. The Golgi fragmentation markedly observed with the neutralized PDFs could be reversed by replacing either the media with a neutral-pH medium or a mixture of PDF and PD effluent (PDF) in a gradient manner mimicking clinical conditions. Furthermore, although weaker than pH effect, notable effects of other PDF-related factors were also observed after 30-min exposure to pH-adjusted PDFs. Lastly, the results of studies conducted using MAPK/SAPK inhibitors indicated that the mechanism underlying the Golgi fragmentation described here differs from that associated with the fragmentation that occurs at the G2/M checkpoint in the cell cycle. We conclude that Golgi fragmentation is suitable for rapid biocompatibility assessment of PDF not only because of its strong pH dependence but also because the fragmentation is recognizably affected by PDF constituents., (© 2018 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP- and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering., (© 2018 Sobajima et al.)

Neutral sphingomyelinase smpd3 is most abundantly expressed in neurons of brain. The function of SMPD3 has remained elusive. Here, we report a pathogenetic nexus between absence of SMPD3 in the Golgi compartment (GC) of neurons of the smpd3-/- mouse brain, inhibition of Golgi vesicular protein transport and progressive cognitive impairment. Absence of SMPD3 activity in the Golgi sphingomyelin cycle impedes remodeling of the lipid bilayer, essential for budding and multivesicular body formation. Importantly, we show that inhibition of the Golgi vesicular protein transport causes accumulation of neurotoxic proteins APP, Aβ and phosphorylated Tau, dysproteostasis, unfolded protein response, and apoptosis, which ultimately manifests in progressive cognitive decline, similar to the pathognomonic signatures of familial and sporadic forms of Alzheimer´s disease. This discovery might contribute to the search for other primary pathogenic mechanisms, which link perturbed lipid bilayer structures and protein processing and transport in the neuronal Golgi compartment and neurodegeneration and cognitive deficits.

Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24 h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope. At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.Key words: Golgi morphology, N-ethylmaleimide-sensitive factor (NSF), Arabidopsis thaliana.

The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field., (© 2018. Published by The Company of Biologists Ltd.)

Mixed-linkage (1,3;1,4)-β-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-β-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other researchers. We found that no progeny viruses were produced by transfection of fetal equine kidney cells and rabbit kidney (RK-13) cells with the UL11 null mutant and truncation mutant BAC DNAs. However, mutant viruses were generated after transfection of RK13-UL11 cells constitutively expressing EHV-1 UL11 with the mutant BAC DNAs. In conclusion, UL11 of EHV-1 Ab4p is essential for replication in cultured cells.

In vertebrates, individual Golgi stacks are joined into a compact ribbon structure; however, the relevance of a ribbon structure has been elusive. Here, we exploit the finding that the membrane tether of the trans -Golgi network, GCC88 (encoded by GCC1 ), regulates the balance between Golgi mini-stacks and the Golgi ribbon. Loss of Golgi ribbons in stable cells overexpressing GCC88 resulted in compromised mechanistic target of rapamycin (mTOR) signaling and a dramatic increase in LC3-II-positive autophagosomes, whereas RNAi-mediated depletion of GCC88 restored the Golgi ribbon and reduced autophagy. mTOR was absent from dispersed Golgi mini-stacks whereas recruitment of mTOR to lysosomes was unaffected. We show that the Golgi ribbon is a site for localization and activation of mTOR, a process dependent on the ribbon structure. We demonstrate a strict temporal sequence of fragmentation of Golgi ribbon, loss of Golgi mTOR and subsequent increased autophagy. Golgi ribbon fragmentation has been reported in various neurodegenerative diseases and we demonstrate the potential relevance of our findings in neuronal cells using a model of neurodegeneration. Overall, this study highlights a role for the Golgi ribbon in pathways central to cellular homeostasis.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Ultrastructure of plasma cells in Harderian gland was investigated using the transmission electron microscopy. For this research, we examined the glands of 32 laying hens collected at 1, 7, 20 and 40 days and 4, 6, 8 and 12 months of the birds' ages. The research showed that the stroma of the gland contains a large number of lymphocytes and plasma cells. Most of the plasma cells are mature, but morphologically do not show productive activity. Only some individual plasma cells, situated under the secretory epithelium of primary and secondary ducts, have extremely dilated cisternae of rough endoplasmic reticulum which contain moderately dense, granular material. The morphology of these cells indicates that they are in active stage of immunoglobulin production. Also, we identified plasma cells with two types of Russell bodies. One type of these bodies was small, round or oval, while the other had irregular, angular shape. It was noted that one plasma cell never contains both type of Russell bodies at the same time. These cells were often affected by apoptosis. Among them, in deeper part of the stroma, were situated the small plasmablast cells., (© 2017 Blackwell Verlag GmbH.)