Search

The cytoskeleton is a master organizer of the cellular cortex and membrane trafficking and therefore plays a crucial role in apico–basal polarity. Septins form a family of GTPases that assemble into non-polar filaments, which bind to membranes and recruit cytoskeletal elements such as microtubules and actin using their polybasic (PB) domains, to perform their broad biological functions. Nevertheless, the role of septins and the significance of their membrane-binding ability in apico–basal polarity remains under-investigated. Here, using 3D cultures, we demonstrated that septin 9 localizes to the basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing β-catenin at BM and increasing transforming growth factor (TGFβ) and Epithelial–Mesenchymal Transition (EMT) markers. Similar effects were observed after deleting its two PB domains. The mutant became cytoplasmic and apical. The cysts with an inverted polarity phenotype displayed an invasive phenotype, with src and cortactin accumulating at the peripheral membrane. The inhibition of TGFβ-receptor and RhoA rescued the polarized phenotype, although the cysts from overexpressed septin 9 overgrew and presented a filled lumen. Both phenotypes corresponded to tumor features. This suggests that septin 9 expression, along with its assembly through the two PB domains, is essential for establishing and maintaining apico–basal polarity against tumor development.

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

International audience; Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose Core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of Core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of Core, which subsequently relocalizes to contiguous ER regions. During this motion, Core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of Core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

During organogenesis, epithelial cells can give rise to mesenchymal cells through epithelial-mesenchymal transition. The reverse process, mesenchymal-epithelial transition (MET), can similarly generate epithelial cells. Transitions between epithelial and mesenchymal states are also critical for the induction of pluripotent stem cells from somatic cells. This Review discusses the relatively less characterized process of MET, focusing on the genesis of apicobasal cell polarity and exploring the roles of MET in development and reprogramming.

Chronic hepatitis C virus (HCV) infection often leads to fibrosis and chronic hepatitis, then cirrhosis and ultimately hepatocellular carcinoma (HCC). The processes of the HVC life cycle involve intimate interactions between viral and host cell proteins and lipid metabolism. However, the molecules and mechanisms involved in this tripartite interaction remain poorly understood. Herein, we show that the infection of HCC-derived Huh7.5 cells with HCV promotes upregulation of the protein inhibitor of activated STAT1 (PIAS1). Reciprocally, PIAS1 regulated the expression of HCV core protein and HCV-induced LD accumulation and impaired HCV replication. Furthermore, PIAS1 controlled HCV-promoted septin 9 filament formation and microtubule polymerization. Subsequently, we found that PIAS1 interacted with septin 9 and controlled its assembly on filaments, which thus affected septin 9-induced lipid droplet accumulation. Taken together, these data reveal that PIAS1 regulates the accumulation of lipid droplets and offer a meaningful insight into how HCV interacts with host proteins.

International audience; Septins are a family of GTP-binding proteins that assemble into non-polar filaments which can be recruited to negatively charged membranes and serve as a scaffold to recruit cytosolic proteins and cytoskeletal elements such as microtubules and actin so that they can perform their important biological functions. Human septins consist of four groups, each with 13 members, and filaments formation usually involve members from each group in specific positions. However, little is known about the molecular mechanisms that drive the binding of septins to membranes and its importance to their biological functions. Here we have built a protein-protein interaction (PPI) network around human septins and highlighted the connections with 170 partners. Functional enrichment by inference of the network of septins and their partners revealed their participation in functions consistent with some of the roles described for septins, including cell cycle, cell division and cell shape, but we also identified septin partners in these functions that had not previously been described. Interestingly, we identified important and multiple connections between septins and mRNA processing and their export from the nucleus. Analysis of the enrichment of gene ontology cellular components highlighted some important interactions between molecules involved in the spliceosome with septin 2 and septin 7 in particular. RNA splicing regulates gene expression, and through it, cell fate, development and physiology. Mutations in components of the in the splicing machinery is linked to several diseases including cancer, thus taken together, the different analyses presented here open new perspectives to elucidate the pathobiological role of septins.

Summary Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology., Graphical Abstract, Highlights • Two polybasic domains mediate septin 9 interactions with PIs • Human septins have amphipathic helices suitable for membrane binding • Septin 9 polybasic domains mediate the formation of septin higher-order structures • The mutation or depletion of septin polybasic domains induces Golgi fragmentation, Membrane Architecture; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology

Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains contact adjacent cells and apical domains face the exterior lumen. Each membrane domain is endowed with a specific macromolecular composition that constitutes the functional identity of that domain. Defects in apical-basal plasma membrane polarity altogether or more subtle defects in the composition of either apical or basal plasma membrane domain can give rise to severe diseases. Lipids are the main component of cellular membranes and mechanisms that control their polarized distribution in epithelial cells are emerging. In particular sphingolipids and phosphatidylinositol lipids have taken center stage in the organization of the apical and basolateral plasma membrane domain. This short review article discusses mechanisms that contribute to the polarized distribution of lipids in epithelial cells.

During organogenesis, epithelial cells can give rise to mesenchymal cells through epithelial-mesenchymal transition. The reverse process, mesenchymal-epithelial transition (MET), can similarly generate epithelial cells. Transitions between epithelial and mesenchymal states are also critical for the induction of pluripotent stem cells from somatic cells. This Review discusses the relatively less characterized process of MET, focusing on the genesis of apicobasal cell polarity and exploring the roles of MET in development and reprogramming.

Septins belong to a family of GTP‐binding proteins that bind to phosphoinositides (PIs) by a polybasic domain (PB1). We reported that the deletion of PB1 in septin 9 markedly reduced, its binding to PIs and its assembly. Here, using homology modelling, we reveal the presence of another polybasic domain (PB2) conserved among the different human septins. PB1 and PB2 from two distinct molecules form an extended basic cluster in the NC interface of the filament. Importantly binding of septin 9 to PIs through PB1 and PB2 is required for recruitment of microtubules to Golgi stacks and their assembly. Knockdown of septin 9 recapitulates these effects and affects nucleus/centrosomal positioning and protein secretion. Thus septin 9 could be considered as a crucial regulator of events require for membrane trafficking and organelle morphology.

International audience; Lumen formation during epithelial morphogenesis requires the creation of a luminal space at cell interfaces named apical membrane-initiation sites (AMISs). This is dependent upon integrated signaling from mechanical and biochemical cues, vesicle trafficking, cell division, and processes tightly coupled to ciliogenesis. Deciphering relationships between polarity determinants and lumen or cilia generation remains a fundamental issue. Here, we report that Src homology 2 domain-containing inositol 5-phosphatase 2 (SHIP2), a basolateral determinant of polarity, regulates RhoA-dependent actin contractility and cell division to form AMISs. SHIP2 regulates mitotic spindle alignment. SHIP2 is expressed in G1 phase, whereas Aurora A kinase is enriched in mitosis. SHIP2 binds Aurora A kinase and the scaffolding protein HEF1 and promotes their basolateral localization at the expense of their luminal expression connected with cilia resorption. Furthermore, SHIP2 expression increases cilia length. Thus, our findings offer new insight into the relationships among basolateral proteins, lumen generation, and ciliogenesis.

The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway., The accumulation of lipid droplets is often observed in hepatitis C virus infection, but the mechanism of their formation is not known. Here the authors show that septin 9 expression is increased in infected livers, and a septin 9/phosphatidylinositol 5-phosphate signalling pathway regulates the growth of lipid droplets.

Many glandular epithelia such as the GI tract are composed of a sheet of cells polarised along their apicobasal axes with the apical membrane facing the lumen of the tube and the basolateral membrane binding to neighbouring cells and the basal extracellular matrix (ECM). Generation of these two membrane domains with distinct cellular macromolecular contents (including proteins and lipids) provides the basic building blocks that support tissue architecture and their various functions.1 The apicobasal polarisation of epithelial cells is a complex and multistage programme controlled by a network of proteins and lipids. It is initiated by cues from the ECM and cell–cell contacts that trigger changes to the cytoskeleton and hence the polarised organisation of endosomal trafficking that sort and deliver proteins and lipids to the apical and basolateral membrane domains. These events lead to the establishment of cell–cell junctional complexes comprising tight and adherens junctions2 (figure 1A). The establishment of polarity also requires the proper assembly and localisation of a set of evolutionarily conserved polarity proteins. These include the Par3/Par6/atypical protein kinase C (aPKC) and Crumbs/protein associated with Caenorhabditis elegans Lin-7 protein (PALS)/PALS1-associated TJ protein (PATJ) complexes that are mainly found apically. The Scribble/discs large (Dlg)/lethal giant larvae (Lgl) complex is located on the lateral surface of the cell2 (figure 1A). Another Par protein is liver kinase b1 (Lkb1, or Par4), which phosphorylates and activates a family of AMP-activated protein kinases to regulate cell growth, metabolism and polarity.1 Class I isoforms of phosphoinositide 3-kinases (PI3K)3 and their antagonising phosphatases phosphatase and tensin homologue4 (PTEN)) and SH2 domain containing inositol 5-phosphatase 25 (SHIP2) are crucial determinants of apicobasal polarity (figure 1A). Figure 1 Disruption of apicobasal polarity in cancer. (A) Major determinants of polarity in epithelial cells. (1) Normal simple glandular epitheliums …

Infection by hepatitis C virus (HCV) is a major public-health problem. Chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. The life cycle of HCV depends on the host cell machinery and involves intimate interaction between viral and host proteins. However, the role of host proteins in the life cycle of HCV remains poorly understood. Here, we identify the small ubiquitin-related modifier (SUMO1) as a key host factor required for HCV replication. We performed a series of cell biology and biochemistry experiments using the HCV JFH-1 (Japanese fulminate hepatitis 1) genotype 2a strain, which produces infectious particles and recapitulates all the steps of the HCV life cycle. We observed that SUMO1 is upregulated in Huh7.5 infected cells. Reciprocally, SUMO1 was found to regulate the expression of viral core protein. Moreover, knockdown of SUMO1 using specific siRNA influenced the accumulation of lipid droplets and reduced HCV replication as measured by qRT-PCR. Thus, we identify SUMO1 as a key host factor required for HCV replication. To our knowledge, this is the first report showing that SUMO1 regulates lipid droplets in the context of viral infection. Our report provides a meaningful insight into how HCV replicates and interacts with host proteins and is of significant importance for the field of HCV and RNA viruses.