The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Fluidic shear stress alters clathrin dynamics and vesicle formation in endothelial cells.

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs. It remains elusive how ECs maintain CME in these biophysically unfavorable conditions. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to show that endocytic dynamics are similar between statically cultured human umbilical vein endothelial cells (HUVECs) and fibroblast-like Cos-7 cells. Here, we asked whether biophysical stresses generated by blood flow influence CME. We used our data processing platform-DrSTAR-to examine if clathrin dynamics are altered in HUVECs after experiencing fluidic shear stress (FSS). We found that HUVECs cultivated under a physiological level of FSS had increased clathrin dynamics compared with static controls. FSS increased both clathrin-coated vesicle formation and nonproductive flat clathrin lattices by 2.3-fold and 1.9-fold, respectively. The curvature-positive events had significantly delayed curvature initiation relative to clathrin recruitment in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and clathrin-coated vesicle formation can be modulated by the local physiological environment and represent an important regulatory mechanism.

A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication.

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Cystin is required for maintaining fibrocystin (FPC) levels and safeguarding proteome integrity in mouse renal epithelial cells: A mechanistic connection between the kidney defects in cpk mice and human ARPKD.

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in congenital polycystic kidney (cpk) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r-cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

INTRODUCTION: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. METHODS: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. RESULTS: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. CONCLUSION: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles.

Enterovirus replication requires the cellular protein GBF1, a guanine nucleotide exchange factor for small Arf GTPases. When activated, Arfs associate with membranes, where they regulate numerous steps of membrane homeostasis. The requirement for GBF1 implies that Arfs are important for replication, but which of the different Arfs function(s) during replication remains poorly understood. Here, we established cell lines expressing each of the human Arfs fused to a fluorescent tag and investigated their behavior during enterovirus infection. Arf1 was the first to be recruited to the replication organelles, where it strongly colocalized with the viral antigen 2B and mature virions but not double-stranded RNA. By the end of the infectious cycle, Arf3, Arf4, Arf5, and Arf6 were also concentrated on the replication organelles. Once on the replication membranes, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that in infected cells they do not actively cycle between GTP- and GDP-bound states. Only the depletion of Arf1, but not other class 1 and 2 Arfs, significantly increased the sensitivity of replication to GBF1 inhibition. Surprisingly, depletion of Arf6, a class 3 Arf, normally implicated in plasma membrane events, also increased the sensitivity to GBF1 inhibition. Together, our results suggest that GBF1-dependent Arf1 activation directly supports the development and/or functioning of the replication complexes and that Arf6 plays a previously unappreciated role in viral replication. Our data reveal a complex pattern of Arf activation in enterovirus-infected cells that may contribute to the resilience of viral replication in different cellular environments.IMPORTANCE Enteroviruses include many known and emerging pathogens, such as poliovirus, enteroviruses 71 and D68, and others. However, licensed vaccines are available only against poliovirus and enterovirus 71, and specific anti-enterovirus therapeutics are lacking. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, replication organelles. Here, we investigated the roles of small Arf GTPases during enterovirus infection. Arfs control distinct steps in intracellular membrane traffic, and one of the Arf-activating proteins, GBF1, is a cellular factor required for enterovirus replication. We found that all Arfs expressed in human cells, including Arf6, normally associated with the plasma membrane, are recruited to the replication organelles and that Arf1 appears to be the most important Arf for enterovirus replication. These results document the rewiring of the cellular membrane pathways in infected cells and may provide new ways of controlling enterovirus infections.

JAGN1, tetraspanins, and Erv proteins: is common topology indicative of common function in cargo sorting?

The endoplasmic reticulum protein Jagunal (JAGN1) was first identified as a requirement for Drosophila melanogaster oocyte development. Subsequent studies in human patients linked mutations in JAGN1 to severe congenital neutropenia, as well as a broad range of additional symptoms, suggesting that JAGN1 function is required in many tissues. Moreover, JAGN1 orthologs are found throughout animal and plant phylogeny, suggesting that JAGN1 supports fundamental cellular processes not restricted to egg development or neutrophil function. JAGN1 lacks sequence similarity or recognizable domains other than a coatomer protein complex I-binding motif, and its cellular function is currently unknown. JAGN1 shares a tetraspanning membrane topology with two families of known cargo transporters: the tetraspanins and the endoplasmic reticulum vesicle (Erv) proteins. Herein, we discuss the similarities between JAGN1, tetraspanins, and Ervs and, based on those, suggest a role for JAGN1 in facilitating the traffic of cell-restricted and ubiquitously expressed proteins at the endoplasmic reticulum-Golgi interface.

ARF family GTPases with links to cilia.

The ADP-ribosylation factor (ARF) superfamily of regulatory GTPases, including both the ARF and ARF-like (ARL) proteins, control a multitude of cellular functions, including aspects of vesicular traffic, lipid metabolism, mitochondrial architecture, the assembly and dynamics of the microtubule and actin cytoskeletons, and other pathways in cell biology. Considering their general utility, it is perhaps not surprising that increasingly ARF/ARLs have been found in connection to primary cilia. Here, we critically evaluate the current knowledge of the roles four ARF/ARLs (ARF4, ARL3, ARL6, ARL13B) play in cilia and highlight key missing information that would help move our understanding forward. Importantly, these GTPases are themselves regulated by guanine nucleotide exchange factors (GEFs) that activate them and by GTPase-activating proteins (GAPs) that act as both effectors and terminators of signaling. We believe that the identification of the GEFs and GAPs and better models of the actions of these GTPases and their regulators will provide a much deeper understanding and appreciation of the mechanisms that underly ciliary functions and the causes of a number of human ciliopathies.