Calcium-binding Cab45 regulates the polarized apical secretion of soluble proteins in epithelial cells.

Protein secretion is essential for epithelial tissue homoeostasis and therefore has to be tightly regulated. However, while the mechanisms regulating polarized protein sorting and trafficking have been widely studied in the past decade, those governing polarized secretion remain elusive. The calcium manganese pump SPCA1 and the calcium-binding protein Cab45 were recently shown to regulate the secretion of a subset of soluble cargoes in nonpolarized HeLa cells. Interestingly, we demonstrated that in polarized epithelial cells calcium levels in the trans-Golgi network (TGN), controlled by SPCA1, and Cab45 are critical for the apical sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs), a class of integral membrane proteins containing a soluble protein attached to the membrane by the GPI anchor, prompting us to investigate the mechanism regulating the polarized secretion of soluble cargoes. By reducing Cab45 expression level or overexpressing an inactive mutant of SPCA1, we found that Cab45 and calcium levels in the TGN drive the polarized apical secretion of a secretory form of placental alkaline phosphatase, exogenously expressed, and the endogenous soluble protein clusterin/Gp80 in Madin-Darby canine kidney (MDCK) cells. These data highlight the critical role of a calcium-dependent Cab45 mechanism regulating apical exocytosis in polarized MDCK cells.

Persistence of uncontrolled hypertension post-cardiac rehabilitation in stable coronary patients.

In stable coronary heart disease, uncontrolled risk factors are strongly associated with incident myocardial infarction. We analysed the management of hypertension in 746 stable coronary patients recruited between 2005 and 2015 in a single-centre prospective study. Risk factors and pharmacological treatments were documented prior to and immediately after cardiac rehabilitation, and 1 year later. One year post-cardiac rehabilitation, all cardiovascular risk factors were significantly better controlled with the notable exception of hypertension: blood pressure (BP) <140/90 mmHg in 60% of the total population vs 49% (N = 450) of hypertensive patients (20% or 10%, according to the ACC/AHA 2017 or ESH/ESC guidelines, respectively). Of those who had achieved normotension by the end of cardiac rehabilitation, 42% had uncontrolled hypertension again 1 year later; in addition, body weight had increased, while physical activity and antihypertensive drug use had dropped (differences between controlled or uncontrolled hypertension at 1 year post-cardiac rehabilitation, NS). Three factors were correlated with BP elevations: discontinuation of betablockade: +7.9 mmHg; age >65 years: +6.2 mmHg; diabetes mellitus: +7.6 mmHg. Only 48% hypertensive patients were on guideline-recommended antihypertensive polytherapy. Although 28% were still hypertensive post-cardiac rehabilitation, and hypertension remained uncontrolled in 70% 1 year later, 61% antihypertensive prescriptions were not adjusted post-cardiac rehabilitation. One year post-cardiac rehabilitation, hypertension was the only cardiovascular risk factor that had not improved. This can be attributed to three main reasons, all associated with BP elevations: precipitous reduction in betablockade, physicians' inertia when faced with uncontrolled hypertension and lack of adherence to international guidelines.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Actin Assembly around the Shigella-Containing Vacuole Promotes Successful Infection.

The enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterium-containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host CDC42, TOCA-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV dependent on its fatty acyltransferase activity. This represents a unique microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon promotes subsequent invasion steps for successful Shigella infection.

Clustering in the Golgi apparatus governs sorting and function of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid APs attached to the extracellular leaflet of the plasma membrane (PM) via a glycolipid anchor. GPI-APs are commonly associated with cholesterol- and sphingolipid-enriched membrane microdomains. These microdomains help regulating various biological activities, by segregating different proteins and lipids in (nanoscale) membrane compartments. In fibroblasts, GPI-APs form actin- and cholesterol-dependent nanoclusters directly at the PM. In contrast, in polarized epithelial cells GPI-APs cluster in the Golgi apparatus, the major protein-sorting hub for the secretory pathway. Golgi clustering is required for the selective sorting of GPI-APs to the apical PM domain, but also regulates their organization and biological activities at the cell surface. In this review, we discuss recent advances in our understanding of the mechanism of GPI-AP sorting to the apical membrane. We focus on the roles of the protein moiety and lipids in the regulation of the clustering of GPI-APs in the Golgi apparatus.

Organization of GPI-anchored proteins at the cell surface and its physiopathological relevance.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrPC, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells.

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures.

Trafficking and Membrane Organization of GPI-Anchored Proteins in Health and Diseases.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of lipid-anchored proteins attached to the membranes by a glycolipid anchor that is added, as posttranslation modification, in the endoplasmic reticulum. GPI-APs are expressed at the cell surface of eukaryotes where they play diverse vital functions. Like all plasma membrane proteins, GPI-APs must be correctly sorted along the different steps of the secretory pathway to their final destination. The presence of both a glycolipid anchor and a protein portion confers special trafficking features to GPI-APs. Here, we discuss the recent advances in the field of GPI-AP trafficking, focusing on the mechanisms regulating their biosynthetic pathway and plasma membrane organization. We also discuss how alterations of these mechanisms can result in different diseases. Finally, we will examine the strict relationship between the trafficking and function of GPI-APs in epithelial cells.

Golgi sorting regulates organization and activity of GPI proteins at apical membranes.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy.

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting.

To understand the mechanism involved in the apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) we fused to the C-terminus of GFP the GPI-anchor-attachment signal of the folate receptor (FR) or of the prion protein (PrP), two native GPI-anchored proteins that are sorted apically or basolaterally, respectively, in MDCK cells. We investigated the behaviour of the resulting fusion proteins GFP-FR and GFP-PrP by analysing three parameters: their association with DRMs, their oligomerisation and their apical sorting. Strikingly, we found that different GPI-attachment signals differently modulate the ability of the resulting GFP-fusion protein to oligomerise and to be apically sorted. This is probably owing to differences in the GPI anchor and/or in the surrounding lipid microenvironment. Accordingly, we show that addition of cholesterol to the cells is necessary and sufficient to drive the oligomerisation and consequent apical sorting of GFP-PrP, which under control conditions does not oligomerise and is basolaterally sorted.

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells.

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.

Coexpression of wild-type and mutant prion proteins alters their cellular localization and partitioning into detergent-resistant membranes.

Transmissible spongiform encephalopathies (TSEs) are a group of diseases of infectious, sporadic and genetic origin, found in higher organisms and caused by the pathological form of the prion protein. The inheritable subgroup of TSEs is linked to insertional or point mutations in the prion gene prnp, which favour its misfolding and are passed on to offspring in an autosomal-dominant fashion. The large majority of patients with these diseases are heterozygous for the prnp gene, leading to the coexpression of the wild-type (wt) (PrP(C)) and the mutant forms (PrPmut) in the carriers of these mutations. To mimic this situation in vitro, we produced Fischer rat thyroid cells coexpressing PrPwt alongside mutant versions of mouse PrP including A117V, E200K and T182A relevant to the human TSE diseases Gestmann-Sträussler-Scheinker (GSS) disease and familial Creutzfeldt-Jakob disease (fCJD). We found that coexpression of mutant PrP with wt proteins does not affect the glycosylation pattern or the biochemical characteristics of either protein. However, FRET and co-immunoprecipitation experiments suggest an interaction occurring between the wt and mutant proteins. Furthermore, by comparing the intracellular localization and detergent-resistant membrane (DRM) association in single- and double-expressing clones, we found changes in the intracellular/surface ratio and an increased sequestration of both proteins in DRMs, a site believed to be involved in the pathological conversion (or protection thereof) of the prion protein. We, therefore, propose that the mutant forms alter the subcellular localization and the membrane environment of the wt protein in co-transfected cells. These effects may play a role in the development of these diseases.

Emergence of organizer function: a lot of "stuff" involved.

Spemann's Organizer is a critical signaling center for patterning the embryo. It arises during blastula stages through the combined influences of dorsal modifying signals and general mesendoderm inducers. Dorsal modifying signals require the nuclear accumulation of beta-catenin, but how this is initiated remained a mystery until recently. New findings now demonstrate that maternal Wnt11 activates the canonical Wnt signaling pathway and is essential for organizer formation. Furthermore, two of the earliest identified mesendoderm inducers, activin and Vg-1, have now been shown to be required for induction of a fully functional organizer. Finally, while it has been clear for a number of years that the Organizer secretes a cocktail of growth factor antagonists, their necessity for organizer function has been in question. Their requirement has now been demonstrated through a multiple "knockdown" approach in frog embryos. Here, we discuss the impact these recent findings have on our understanding of formation and function of the Organizer.

RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development.

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.

Control of embryonic Xenopus morphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway.

Ras proteins mediate biological responses through various effectors and play a key role in relaying the Fibroblast Growth Factor (FGF) mesoderm induction signal during embryogenesis of the frog, Xenopus laevis. One Ras effector pathway involves the activation of the small G protein Ral. In the present study, we have investigated the role of key components in the Ral branch of FGF and Ras signalling during early Xenopus development. Treatment of animal caps with bFGF, which converts prospective ectoderm to mesoderm, activates Xral. The Ras mutant 12V37G, which can bind to Ral-GDS but not Raf, also activates Xral as well as causing developmental defects and cortical F-actin disassembly. A similar phenotype is induced by Ral-GDS itself. FGF-induced expression of several signature mesodermal genes, by contrast, is independent of Xral signalling. This and other data suggest that the RalB branch of Ras and FGF signalling regulates the actin cytoskeleton and morphogenesis in a transcriptionally independent manner. We also find Xral to be specifically activated in the marginal zone of Xenopus embryos, and find that disruption of the Ral pathway in this region prevents closure of the blastopore during gastrulation. We conclude that Ral signalling is autonomously required by mesodermal cells to effect essential morphogenetic changes during Xenopus gastrulation.