Actin cytoskeleton differently regulates cell surface organization of GPI-anchored proteins in polarized epithelial cells and fibroblasts.

The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.