Distinct changes in endosomal composition promote NLRP3 inflammasome activation.

Inflammasome complexes are pivotal in the innate immune response. The NLR family pyrin domain containing protein 3 (NLRP3) inflammasome is activated in response to a broad variety of cellular stressors. However, a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill defined. Here, we demonstrate that NLRP3 inflammasome activators primarily converge on disruption of endoplasmic reticulum-endosome membrane contact sites (EECS). This defect causes endosomal accumulation of phosphatidylinositol 4-phosphate (PI4P) and a consequent impairment of endosome-to-trans-Golgi network trafficking (ETT), necessary steps for endosomal recruitment of NLRP3 and subsequent inflammasome activation. Lowering endosomal PI4P levels prevents endosomal association of NLRP3 and inhibits inflammasome activation. Disruption of EECS or ETT is sufficient to enhance endosomal PI4P levels, to recruit NLRP3 to endosomes and to potentiate NLRP3 inflammasome activation. Mice with defects in ETT in the myeloid compartment are more susceptible to lipopolysaccharide-induced sepsis. Our study thus identifies a distinct cellular mechanism leading to endosomal NLRP3 recruitment and inflammasome activation.

The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

ER exit sites take the strain.

Cells are able to adapt their growth to external mechanical strain. A recent study by Phuyal et al (2022) has shown that these responses depend on the heterodimerization of two small GTPases.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.

FGF signalling regulates bone growth through autophagy.

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

ER-Golgi membrane contact sites.

Membrane contact sites (MCSs) are sites where the membranes of two different organelles come into close apposition (10-30 nm). Different classes of proteins populate MCSs including factors that act as tethers between the two membranes, proteins that use the MCSs for their function (mainly lipid or ion exchange), and regulatory proteins and enzymes that can act in trans across the MCSs. The ER-Golgi MCSs were visualized by electron microscopists early in the sixties but have remained elusive for decades due to a lack of suitable methodological approaches. Here we report recent progress in the study of this class of MCSs that has led to the identification of their main morphological features and of some of their components and roles. Among these, lipid transfer proteins and lipid exchange have been the most studied and understood so far. However, many unknowns remain regarding their regulation and their role in controlling key TGN functions such as sorting and trafficking as well as their relevance in physiological and pathological conditions.

Cellular assays for drug discovery in genetic disorders of intracellular trafficking.

Intracellular membrane trafficking is essential for organelle biogenesis, structure, and function; the exchange of material between organelles; and communication between the cell and its external environment. Genetic disorders affecting intracellular trafficking can lead to a variety of human diseases, but specific therapies for these diseases are notably lacking. In this article, we focus on how current knowledge about genetic disorders that affect intracellular trafficking can be used to develop strategies for cell-based assays in order to identify drugs using high-content screening approaches.

Ultrasonographic findings of fluid accumulation and hyperechogenic spots suggestive of surgical site infection following laparotomy for colic in horses.

BACKGROUND: The aim of this study was to compare ultrasonographic findings of the ventral midline incision after exploratory laparotomy for colic in horses with and without surgical site infection (SSI). METHODS: Ultrasonographic examination of the surgical wound was performed on postoperative day 5 (D5) and day 10 (D10) to assess the presence of fluid accumulation, suture sinus formation, hyperechogenic spots and fistulous path. Clinical evaluation of the wound was used to classify horses with and without SSI. The accuracy, sensitivity, specificity and positive and negative predictive values of the ultrasonographic findings were then calculated. Multivariable logistic regression analyses were performed with SSI as a dependent variable and age, sex, breed and ultrasonographic findings as independent variables after univariate and collinearity analyses. RESULTS: Twenty-nine of the 84 horses examined had an SSI. Detection of fluid accumulation and hyperechogenic spots increased the odds for SSI at D5 (odds ratio [OR]: 4.99, 95% confidence interval [CI]: 1.53-16.33, p = 0.008; OR: 10.78, 95% CI: 1.75-26.59, p = 0.01, respectively) and D10 (OR: 11.51, 95% CI: 2.39-55.47, p = 0.002; OR: 12.34, 95% CI: 3.45-44.15, p < 0.001, respectively). LIMITATION: Ultrasonographic images were taken only on the longitudinal section. CONCLUSION: Ultrasonographic examination is helpful in evaluating the surgical incision after laparotomy, with the detection of fluid accumulation and hyperechogenic spots surrounding the sutures being strongly related to SSI.

The Golgi apparatus: an organelle with multiple complex functions.

Remarkable advances have been made during the last few decades in defining the organizational principles of the secretory pathway. The Golgi complex in particular has attracted special attention due to its central position in the pathway, as well as for its fascinating and complex structure. Analytical studies of this organelle have produced significant advances in our understanding of its function, although some aspects still seem to elude our comprehension. In more recent years a level of complexity surrounding this organelle has emerged with the discovery that the Golgi complex is involved in cellular processes other than the 'classical' trafficking and biosynthetic pathways. The resulting picture is that the Golgi complex can be considered as a cellular headquarters where cargo sorting/processing, basic metabolism, signalling and cell-fate decisional processes converge.

Regulation and physiology of membrane contact sites.

Membrane contact sites (MCSs) in addition to impacting the functions of membrane-limited organelles also have a role in the spatial and functional organization of cells, tissues and whole organisms. MCSs have been identified between all organelles and the identification of their molecular composition has progressed significantly in recent years. Equally important is how MCSs respond dynamically to physiological stimuli, how this is regulated, and the physiological roles of MCSs in tissues and at the organismal level, an area that still remains relatively unexplored. In the present review, we focus on the regulation of MCSs, considerations of their function at the organismal level, and how mutations of MCS components linked to genetic diseases might inform us about their physiological relevance.

Deregulation of phosphatidylinositol-4-phosphate in the development of amyotrophic lateral sclerosis 8.

Amyotrophic lateral sclerosis 8 (ALS8) is one of a heterogeneous group of progressive neurodegenerative disorders characterized by the death of motor neurons. ALS8 is caused by mutations in VAPB, a protein that acts at multiple membrane contact sites between the endoplasmic reticulum (ER) and almost all other organelles and thus affects functions at diverse cellular locations. One prominent function mediated by VAPB at these sites is lipid exchange, and a recurrent phenotype observed in all models investigating knockout or knockdown of VAPs is a significant increase in the levels of phosphatidylinositol-4-phosphate (PI4P). Here we consider the relevance of this PI4P deregulation in the development of ALS8 that might represent a potential target for therapeutic intervention.

Illuminating the membrane contact sites between the endoplasmic reticulum and the trans-Golgi network.

Membrane contact sites (MCSs) between different organelles have been identified and extensively studied over the last decade. Several classes of MCSs have now well-established roles, although the contacts between the endoplasmic reticulum (ER) and the trans-side of the Golgi network (TGN) have long remained elusive. Until recently, the study of ER-TGN contact sites has represented a major challenge in the field, as a result of the lack of suitable visualization and isolation techniques. Only in the last 5 years has the combination of advanced technologies and innovative approaches permitted the identification of new molecular players and the functions of ER-TGN MCSs that couple lipid metabolism and anterograde transport. Although much has yet to be discovered, it is now established that ER-TGN MCSs control phosphatidyl-4-phosphate homeostasis by coupling the cis and the trans activity of the ER-resident 4-phosphatase Sac1. In this review, we focus on recent advances on the composition and function of ER-TGN MCSs.

The activity of Sac1 across ER-TGN contact sites requires the four-phosphate-adaptor-protein-1.

Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. Here, we show that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. We envision that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit.

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system.

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.

A novel class of small repetitive DNA sequences in Enterococcus faecalis.

The structural organization of Enterococcus faecalis repeats (EFAR) is described, palindromic DNA sequences identified in the genome of the Enterococcus faecalis V583 strain by in silico analyses. EFAR are a novel type of miniature insertion sequences, which vary in size from 42 to 650 bp. Length heterogeneity results from the variable assembly of 16 different sequence types. Most elements measure 170 bp, and can fold into peculiar L-shaped structures resulting from the folding of two independent stem-loop structures (SLSs). Homologous chromosomal regions lacking or containing EFAR sequences were identified by PCR among 20 E. faecalis clinical isolates of different genotypes. Sequencing of a representative set of 'empty' sites revealed that 24-37 bp-long sequences, unrelated to each other but all able to fold into SLSs, functioned as targets for the integration of EFAR. In the process, most of the SLS had been deleted, but part of the targeted stems had been retained at EFAR termini.

PI(4)P homeostasis: Who controls the controllers?

During recent decades, PI(4)P (phosphoinositol-4-phosphate) has been described as a key regulator of a wide range of cellular functions such as organelle biogenesis, lipid metabolism and distribution, membrane trafficking, ion channels, pumps, and transporter activities. In this review we will focus on the multiple mechanisms that regulate PI(4)P homeostasis ranging from those responsible for the spatial distribution of the PI4 kinases and PI(4)P phosphatase to those controlling their enzymatic activity or the delivery/presentation of the substrate, i.e. PI or PI(4)P, to the PI4Ks or PI(4)P phosphatase, respectively. We will also highlight the open questions in the field mainly dealing with the existence and mode of action of PI(4)P sensors that monitor its amount and can act as a rheostat tuning PI(4)P levels in different compartments and adapting them to the different needs of the cell.