Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis.

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage.

The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA-ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc-25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV-3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.

Vps33B is required for delivery of endocytosed cargo to lysosomes.

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events.

A suite of genome-engineered hepatic cells provide novel insights into the spatiotemporal metabolism of APOB and APOB-containing lipoprotein secretion.

AIMS: APOB-containing very-low-density lipoprotein (VLDL) production, secretion, and clearance by hepatocytes is a central determinant of hepatic and circulating lipid levels. Impairment of any of the aforementioned processes is associated with the development of multiple diseases. Despite the discovery of genes and processes that govern hepatic VLDL metabolism, our understanding of the different mechanistic steps involved is far from complete. An impediment to these studies is the lack of tractable hepatocyte-based systems to interrogate and follow APOB in cells, which the current study addresses. METHODS AND RESULTS: To facilitate the cellular study of VLDL metabolism, we generated human hepatic HepG2 and Huh-7 cell lines in which CRISPR/Cas9-based genome engineering was used to introduce the fluorescent protein mNeonGreen into the APOB gene locus. This results in the production of APOB100-mNeon that localizes predominantly to the endoplasmic reticulum (ER) and Golgi by immunofluorescence and electron microscopy imaging. The production and secretion of APOB100-mNeon can be quantitatively followed in medium over time, and results in production of lipoproteins that are taken up via the LDLR pathway. Importantly, the production and secretion of APOB-mNeon is sensitive to established pharmacological and physiological treatments, and to genetic modifiers known to influence VLDL production in humans. As a showcase, we used HepG2-APOBmNeon cells to interrogate ER-associated degradation (ERAD) of APOB. Using a dedicated sgRNA library targeting all established membrane-associated ER-resident E3 ubiquitin ligases led to identification of SYNV1 as the E3 responsible for degradation of poorly-lipidated APOB in HepG2 cells. CONCLUSIONS: In summary, the engineered cells reported here allow the study of hepatic VLDL assembly and secretion, and facilitate spatiotemporal interrogation induced by pharmacologic and genetic perturbations.

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1-4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

Bimodal endocytic probe for three-dimensional correlative light and electron microscopy.

We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications.

Ultrastructural Characteristics of Myocardial Reperfusion Injury and Effect of Selective Intracoronary Hypothermia: An Observational Study in Isolated Beating Porcine Hearts.

In acute myocardial infarction (AMI), myocardial reperfusion injury may undo part of the recovery after revascularization of the occluded coronary artery. Selective intracoronary hypothermia is a novel method aimed at reducing myocardial reperfusion injury, but its presumed protective effects in AMI still await further elucidation. This proof-of-concept study assesses the potential protective effects of selective intracoronary hypothermia in an ex-vivo, isolated beating heart model of AMI. In four isolated Langendorff perfused beating pig hearts, an anterior wall myocardial infarction was created by inflating a balloon in the mid segment of the left anterior descending (LAD) artery. After one hour, two hearts were treated with selective intracoronary hypothermia followed by normal reperfusion (cooled hearts). In the other two hearts, the balloon was deflated after one hour, allowing normal reperfusion (control hearts). Biopsies for histologic and electron microscopic evaluation were taken from the myocardium at risk at different time points: before occlusion (t = BO); 5 minutes before reperfusion (t = BR); and 10 minutes after reperfusion (t = AR). Electron microscopic analysis was performed to evaluate the condition of the mitochondria. Histological analyses included evaluation of sarcomeric collapse and intramyocardial hematoma. Electron microscopic analysis revealed intact mitochondria in the hypothermia treated hearts compared to the control hearts where mitochondria were more frequently damaged. No differences in the prespecified histological parameters were observed between cooled and control hearts at t = AR. In the isolated beating porcine heart model of AMI, reperfusion was associated with additional myocardial injury beyond ischemic injury. Selective intracoronary hypothermia preserved mitochondrial integrity compared to nontreated controls.

Pancreatic ß-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic ß-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in ß-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662* ; VPS41c.1423-2A>G and VPS41R662* ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

Regional differences in recombination hotspots between two chicken populations.

BACKGROUND: Although several genetic linkage maps of the chicken genome have been published, the resolution of these maps is limited and does not allow the precise identification of recombination hotspots. The availability of more than 3.2 million SNPs in the chicken genome and the recent advances in high throughput genotyping techniques enabled us to increase marker density for the construction of a high-resolution linkage map of the chicken genome. This high-resolution linkage map allowed us to study recombination hotspots across the genome between two chicken populations: a purebred broiler line and a broiler x broiler cross. In total, 1,619 animals from the two different broiler populations were genotyped with 17,790 SNPs. RESULTS: The resulting linkage map comprises 13,340 SNPs. Although 360 polymorphic SNPs that had not been assigned to a known chromosome on chicken genome build WASHUC2 were included in this study, no new linkage groups were found. The resulting linkage map is composed of 31 linkage groups, with a total length of 3,054 cM for the sex-average map of the combined population. The sex-average linkage map of the purebred broiler line is 686 cM smaller than the linkage map of the broiler x broiler cross. CONCLUSIONS: In this study, we present a linkage map of the chicken genome at a substantially higher resolution than previously published linkage maps. Regional differences in recombination hotspots between the two mapping populations were observed in several chromosomes near the telomere of the p arm; the sex-specific analysis revealed that these regional differences were mainly caused by female-specific recombination hotspots in the broiler x broiler cross.

The function of GORASPs in Golgi apparatus organization in vivo.

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity.

BACKGROUND: The CLN3 disease spectrum ranges from a childhood-onset neurodegenerative disorder to a retina-only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well-known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. METHODS: Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal-associated membrane protein 1 (LAMP-1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. RESULTS: Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole-shaped LAMP-1 expression, suggesting the use of LAMP-1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP-1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in-depth revealing that intracellular LAMP-1 expression was most pronounced in T-cells of classical-protracted CLN3 disease while it was most pronounced in B-cells of "retina-only" CLN3 disease. CONCLUSION: Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.

Vps3 and Vps8 control integrin trafficking from early to recycling endosomes and regulate integrin-dependent functions.

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.

Ultrastructural Characterization of Membrane Rearrangements Induced by Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) belonging to the α-CoV genus and it causes high mortality in infected sucking piglets, resulting in substantial losses in the farming industry. CoV trigger a drastic reorganization of host cell membranes to promote their replication and egression, but a detailed description of the intracellular remodeling induced by PEDV is still missing. In this study, we examined qualitatively and quantitatively, using electron microscopy, the intracellular membrane reorganization induced by PEDV over the course of an infection. With our ultrastructural approach, we reveal that, as most of CoV, PEDV initially forms double-membrane vesicles (DMVs) and convoluted membranes (CMs), which probably serve as replication/transcription platforms. Interestingly, we also found that viral particles start to form almost simultaneously in both the endoplasmic reticulum and the large virion-containing vacuoles (LVCVs), which are compartments originating from the Golgi, confirming that α-CoV assemble indistinguishably in two different organelles of the secretory pathway. Moreover, PEDV virons appear to have an immature and a mature form, similar to another α-CoV the transmissible gastroenteritis coronavirus (TGEV). Altogether, our study underlies the similarities and differences between the lifecycle of α-CoV and that of viruses belonging to other CoV subfamilies.

Phospho-Rasputin Stabilization by Sec16 Is Required for Stress Granule Formation upon Amino Acid Starvation.

Most cellular stresses induce protein translation inhibition and stress granule formation. Here, using Drosophila S2 cells, we investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, we find that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, we identify Sec16, a component of the endoplasmic reticulum exit site, as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein.

In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation.

PARP catalysed ADP-ribosylation is a post-translational modification involved in several physiological and pathological processes, including cellular stress. In order to visualise both Poly-, and Mono-, ADP-ribosylation in vivo, we engineered specific fluorescent probes. Using them, we show that amino-acid starvation triggers an unprecedented display of mono-ADP-ribosylation that governs the formation of Sec body, a recently identified stress assembly that forms in Drosophila cells. We show that dPARP16 catalytic activity is necessary and sufficient for both amino-acid starvation induced mono-ADP-ribosylation and subsequent Sec body formation and cell survival. Importantly, dPARP16 catalyses the modification of Sec16, a key Sec body component, and we show that it is a critical event for the formation of this stress assembly. Taken together our findings establish a novel example for the role of mono-ADP-ribosylation in the formation of stress assemblies, and link this modification to a metabolic stress.