OCRL1 Deficiency Affects the Intracellular Traffic of ApoER2 and Impairs Reelin-Induced Responses.

Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.

Visual EMDR stimulation mitigates acute varied stress effects on morphology of hippocampal neurons in male Wistar rats.

Introduction: Stress is a pervasive health concern known to induce physiological changes, particularly impacting the vulnerable hippocampus and the morphological integrity of its main residing cells, the hippocampal neurons. Eye Movement Desensitization and Reprocessing (EMDR), initially developed to alleviate emotional distress, has emerged as a potential therapeutic/preventive intervention for other stress-related disorders. This study aimed to investigate the impact of Acute Variable Stress (AVS) on hippocampal neurons and the potential protective effects of EMDR. Methods: Rats were exposed to diverse stressors for 7 days, followed by dendritic morphology assessment of hippocampal neurons using Golgi-Cox staining. Results: AVS resulted in significant dendritic atrophy, evidenced by reduced dendritic branches and length. In contrast, rats receiving EMDR treatment alongside stress exposure exhibited preserved dendritic morphology comparable to controls, suggesting EMDR's protective role against stressinduced dendritic remodeling. Conclusions: These findings highlight the potential of EMDR as a neuroprotective intervention in mitigating stress-related hippocampal alterations.

Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.

Myristoylated GRASP55 dimerizes in the presence of model membranes.

The Golgi Reassembly and Stacking Proteins (GRASPs) are engaged in various functions within the cell, both in unconventional secretion mechanisms and structuring and organizing the Golgi apparatus. Understanding their specific role in each situation still requires more structural and functional data at the molecular level. GRASP55 is one of the GRASP members in mammals, anchored to the membrane via the myristoylation of a Gly residue at its N-terminus. Therefore, co-translational modifications, such as myristoylation, are fundamental when considering a strategy to obtain detailed information on the interactions between GRASP55 and membranes. Despite its functional relevance, the N-terminal myristoylation has been underappreciated in the studies reported to date, compromising the previously proposed models for GRASP-membrane interactions. Here, we investigated the synergy between the presence of the membrane and the formation of oligomeric structures of myristoylated GRASP55, using a series of biophysical techniques to perform the structural characterization of the lipidated GRASP55 and its interaction with biological lipid model membranes. Our data fulfill an unexplored gap: the adequate evaluation of the presence of lipidations and lipid membranes on the structure-function dyad of GRASPs.Communicated by Ramaswamy H. Sarma.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia.

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

GoSAMTs are required for pectin methyl-esterification and mucilage release in seed coat epidermal cells.

Introduction: GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. Methods: To determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. Results: We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of "egg-box" structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. Discussion: The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants.

Facial nerve axotomy induces morphological changes in hippocampal pyramidal neurons.

Facial nerve injury in rats have been widely used to study functional and structural changes that occur in the injured motoneurons and other central nervous system structures related with sensorimotor processing. A decrease in long-term potentiation of hippocampal CA3-to-CA1 commissural synapse has recently been reported related to this peripheral injury. Additionally, it has been found increased corticosterone plasmatic levels, impairment in spatial memory consolidation, and hippocampal microglial activation in animals with facial nerve axotomy. In this work, we analyzed the neuronal morphology of hippocampal CA1 and CA3 pyramidal neurons in animals with either reversible or irreversible facial nerve injury. For this purpose, brain tissues of injured animals sacrificed at different postlesion times, were stained with the Golgi-Cox method and compared with control brains. It was found that both reversible and irreversible facial nerve injury-induced significant decreases in dendritic tree complexity, dendritic length, branch points, and spine density of hippocampal neurons. However, such changes' timing varied according to hippocampal area (CA1 vs. CA3), dendritic area (apical vs. basal), and lesion type (reversible vs. irreversible). In general, the observed changes were transient when animals had the possibility of motor recovery (reversible injury), but perdurable if the recovery from the lesion was impeded (irreversible injury). CA1 apical and CA3 basal dendritic tree morphology were more sensible to irreversible injury. It is concluded that facial nerve injury induced significant changes in hippocampal CA1 and CA3 pyramidal neurons morphology, which could be related to LTP impairments and microglial activation in the hippocampal formation, previously described.

Clinical implications of Golgi protein 73 and granulocyte colony-stimulating factor and their related factors in children with bronchopneumonia

Abstract Objective: To investigate the clinical implications of Golgi glycoprotein 73 (GP73) and granulocyte colony-stimulating factor (G-CSF) in children with bronchopneumonia (BP). Methods: Seventy-two children with BP (observation group) and 81 healthy children (control group) consecutively brought to the present study's hospital between June 2019 and October 2020 were enrolled. GP73 and G-CSF levels were determined to analyze their diagnostic value for pediatric BP. High-sensitivity C-reactive protein (hs-CRP) was also measured. The clinical implications of GP73 and G-CSF in pediatric BP complicated with respiratory failure and their connections with the inflammatory response were discussed. Results: GP73 and G-CSF levels were remarkably higher in the observation group (p< 0.05). The sensitivity and specificity of combined detection (GP73+G-CSF) in predicting pediatric BP were 72.22% and 86.42%, respectively (p < 0.001 ). GP73 and G-CSF, which are closely related to X-ray classification and complications in the observation group, decreased after treatment and were positively correlated with hs-CRP (p < 0.05), especially in children complicated with respiratory failure. Regression analysis identified the independence of the course of the disease, hs-CRP, X-ray classification, GP73, and G-CSF as influencing factors of respiratory failure in children with BP (p < 0.05). Conclusion: GP73 and G-CSF, with elevated levels in children with BP, are strongly linked to disease progression and are independent influencing factors of respiratory failure, which may be the key to diagnosing and treating pediatric BP in the future.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

Clinical implications of Golgi protein 73 and granulocyte colony-stimulating factor and their related factors in children with bronchopneumonia.

OBJECTIVE: To investigate the clinical implications of Golgi glycoprotein 73 (GP73) and granulocyte colony-stimulating factor (G-CSF) in children with bronchopneumonia (BP). METHODS: Seventy-two children with BP (observation group) and 81 healthy children (control group) consecutively brought to the present study's hospital between June 2019 and October 2020 were enrolled. GP73 and G-CSF levels were determined to analyze their diagnostic value for pediatric BP. High-sensitivity C-reactive protein (hs-CRP) was also measured. The clinical implications of GP73 and G-CSF in pediatric BP complicated with respiratory failure and their connections with the inflammatory response were discussed. RESULTS: GP73 and G-CSF levels were remarkably higher in the observation group (p < 0.05). The sensitivity and specificity of combined detection (GP73+G-CSF) in predicting pediatric BP were 72.22% and 86.42%, respectively (p < 0.001). GP73 and G-CSF, which are closely related to X-ray classification and complications in the observation group, decreased after treatment and were positively correlated with hs-CRP (p < 0.05), especially in children complicated with respiratory failure. Regression analysis identified the independence of the course of the disease, hs-CRP, X-ray classification, GP73, and G-CSF as influencing factors of respiratory failure in children with BP (p < 0.05). CONCLUSION: GP73 and G-CSF, with elevated levels in children with BP, are strongly linked to disease progression and are independent influencing factors of respiratory failure, which may be the key to diagnosing and treating pediatric BP in the future.

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases.

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and ß3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/ß3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.

Neuronopathic Gaucher disease: Beyond lysosomal dysfunction.

Gaucher disease (GD) is an inherited disorder caused by recessive mutations in the GBA1 gene that encodes the lysosomal enzyme ß-glucocerebrosidase (ß-GC). ß-GC hydrolyzes glucosylceramide (GluCer) into glucose and ceramide in the lysosome, and the loss of its activity leads to GluCer accumulation in different tissues. In severe cases, enzymatic deficiency triggers inflammation, organomegaly, bone disease, and neurodegeneration. Neuronopathic Gaucher disease (nGD) encompasses two different forms of the disease, characterized by chronic or acute damage to the central nervous system (CNS). The cellular and molecular studies that uncover the pathological mechanisms of nGD mainly focus on lysosomal dysfunction since the lysosome is the key organelle affected in GD. However, new studies show alterations in other organelles that contribute to nGD pathology. For instance, abnormal accumulation of GluCer in lysosomes due to the loss of ß-GC activity leads to excessive calcium release from the endoplasmic reticulum (ER), activating the ER-associated degradation pathway and the unfolded protein response. Recent evidence indicates mitophagy is altered in nGD, resulting in the accumulation of dysfunctional mitochondria, a critical factor in disease progression. Additionally, nGD patients present alterations in mitochondrial morphology, membrane potential, ATP production, and increased reactive oxygen species (ROS) levels. Little is known about potential dysfunction in other organelles of the secretory pathway, such as the Golgi apparatus and exosomes. This review focuses on collecting evidence regarding organelle dysfunction beyond lysosomes in nGD. We briefly describe cellular and animal models and signaling pathways relevant to uncovering the pathological mechanisms and new therapeutic targets in GD.

Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments.

One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-ß (Aß) peptides in extracellular plaques. The direct precursor of Aß is the carboxyl-terminal fragment ß (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aß. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aß release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking.

Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.

Neural organization of the third optic neuropil, the lobula, in the highly visual semiterrestrial crab Neohelice granulata.

The visual neuropils (lamina, medulla, and lobula complex) of malacostracan crustaceans and hexapods have many organizational principles, cell types, and functional properties in common. Information about the cellular elements that compose the crustacean lobula is scarce especially when focusing on small columnar cells. Semiterrestrial crabs possess a highly developed visual system and display conspicuous visually guided behaviors. In particular, Neohelice granulata has been previously used to describe the cellular components of the first two optic neuropils using Golgi impregnation technique. Here, we present a comprehensive description of individual elements composing the third optic neuropil, the lobula, of that same species. We characterized a wide variety of elements (140 types) including input terminals and lobula columnar, centrifugal, and input columnar elements. Results reveal a very dense and complex neuropil. We found a frequently impregnated input element (suggesting a supernumerary cartridge representation) that arborizes in the third layer of the lobula and that presents four variants each with ramifications organized following one of the four cardinal axes suggesting a role in directional processing. We also describe input elements with two neurites branching in the third layer, probably connecting with the medulla and lobula plate. These facts suggest that this layer is involved in the directional motion detection pathway in crabs. We analyze and discuss our findings considering the similarities and differences found between the layered organization and components of this crustacean lobula and the lobula of insects.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

In vivo observation of amyloid-like fibrils produced under stress.

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended ß-sheets of amyloids is here used to track fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.