Expression de l'Annexine A2et partenaires pendant la différenciation des cellules épithéliales

International audienceThe members of the annexin family of calcium-and phospholipid-binding proteins participate in different cellular processes. Annexin A2 binds to S100A10 forming a functional heterotetrameric protein that has been involved in many cellular functions such as exocytosis, endocytosis, cell junction formation and actin cytoskeleton dynamics. Herein, we studied annexin A2 cellular movements and looked for its partners during epithelial cell differentiation. By using immunofluorescence, mass spectrometry and western blot analyses after S100A10 affinity column separation, we identified several annexin A2-S100A10 partner candidates. The association of putative annexin A2-S100A10 partner candidates obtained by MS after column affinity was validated by immunofluorescence and sucrose density gradient separation. The results show that three proteins were clearly associated to AnxA2: E-cadherin, actin and caveolin 1. Overall the data show that annexin A2 is able to associate to molecular complexes containing actin, caveolin 1 and flotillin 2 before epithelial differentiation and to complexes containing E-Cadherin, actin and caveolin 1, but not flotillin 2 after cell differentiation. The results indicate that actin, caveolin 1 and E-cadherin are the principal 2 protein partners of annexin A2 in epithelial cells and that the serine phosphorylation of the N-terminal domain does not play an essential role during epithelial cell differentiation.Les membres de la famille des annexines, des protéines liant le calcium et les phospholipides, participent à différentsprocessus cellulaires. L’annexine A2 se lie à S100A10, formant une protéine hétérotétramérique fonctionnelle qui a été impliquéedans plusieurs fonctions cellulaires telles que l’exocytose, l’endocytose, la formation des jonctions cellulaires et la dynamique ducytosquelette. Les auteurs ont étudié ici les mouvements cellulaires de l’annexine A2 et recherché ses partenaires durant ladifférenciation des cellules épithéliales. À l’aide de l’immunofluorescence, de la spectrométrie de masse et des analyses detransferts « western » après séparation sur une colonne d’affinité S100A10, ils ont identifié plusieurs candidats partenaires del’annexine A2–S100A10. L’association de candidats partenaires présumés de l’annexine A2–S100A10 obtenus par SM après lacolonne d’affinité a été validée par immunofluorescence et séparation sur un gradient de densité de sucrose. Les résultatsmontrent que trois protéines sont clairement associées à l’annexine A2 : la E-cadhérine, l’actine et la cavéoline 1. Globalement,les données montrent que l’annexine A2 peut s’associer à des complexes moléculaires comprenant l’actine, la cavéoline et laflotilline 2 avant la différenciation épithéliale et à des complexes comprenant la E-cadhérine, l’actine et la cavéoline 1, mais pasla flotilline 2, après la différenciation cellulaire. Les résultats indiquent que l’actine, la cavéoline et la E-cadhérine constituent lesprincipaux partenaires protéiques de l’annexine A2 dans les cellules épithéliales et la phosphorylation des sérines du domaineN-terminal ne joue pas un rôle essentiel durant la différenciation des cellules épithéliales

Basic cell penetrating peptides induce plasma membrane positive curvature, lipid domain separation and protein redistribution

International audienceBasic cell penetrating peptides are tools for molecular cellular internalization of non membrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organisation. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

International audienceCellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization. 2 Keywords: Membrane invagination/ metabolic energy/ penetratin/ penetrating peptide/ physical endocytosis/ self-induced endocytosis

Nuclear annexin II negatively regulates growth of LNCaP cells and substitution of ser 11 and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II

BACKGROUND: Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II. RESULTS: Annexin II localized in the nucleus is phosphorylated, and the appearance of nuclear phosphorylated annexin II is cell cycle dependent, indicating that phosphorylation may play a role in nuclear entry, retention or export of annexin II. By exogenous expression of annexin II in the annexin II-null LNCaP cells, we show that wild-type annexin II is excluded from the nucleus, whereas the NES mutant annexin II localizes in both the nucleus and cytoplasm. Nuclear retention of annexin II results in reduced cell proliferation and increased doubling time of cells. Expression of annexin II, both wild type and NES mutant, causes morphological changes of the cells. By site-specific substitution of glutamic acid in the place of serines 11 and 25 in the N-terminus, we show that simultaneous phosphorylation of both serines 11 and 25, but not either one alone, prevents nuclear localization of annexin II. CONCLUSION: Our data show that nuclear annexin II is phosphorylated in a cell cycle-dependent manner and that substitution of serines 11 and 25 inhibit nuclear entry of annexin II. Aberrant accumulation of nuclear annexin II retards proliferation of LNCaP cells

The Homeodomain Derived Peptide Penetratin Induces Curvature of Fluid Membrane Domains

BACKGROUND:Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS:Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. CONCLUSIONS/SIGNIFICANCE:The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that the membrane targets for these molecules are preferentially the fluid membrane domains and that the mechanism involves the induction of membrane negative curvature. Consequences on cellular uptake are discussed

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids

Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial.In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β–sheet contributions.These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution

Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Annexin A2 expression and partners during epithelial cell differentiation

The members of the annexin family of calcium- and phospholipid-binding proteins participate in different cellular processes. Annexin A2 binds to S100A10 forming a functional heterotetrameric protein that has been involved in many cellular functions such as exocytosis, endocytosis, cell junction formation and actin cytoskeleton dynamics. Herein, we studied annexin A2 cellular movements and looked for its partners during epithelial cell differentiation. By using immunofluorescence, mass spectrometry and western blot analyses after S100A10 affinity column separation, we identified several annexin A2-S100A10 partner candidates. The association of putative annexin A2-S100A10 partner candidates obtained by MS after column affinity was validated by immunofluorescence and sucrose density gradient separation. The results show that three proteins were clearly associated to AnxA2: E-cadherin, actin and caveolin 1. Overall the data show that annexin A2 is able to associate to molecular complexes containing actin, caveolin 1 and flotillin 2 before epithelial differentiation and to complexes containing E-Cadherin, actin and caveolin 1, but not flotillin 2 after cell differentiation. The results indicate that actin, caveolin 1 and E-cadherin are the principal protein partners of annexin A2 in epithelial cells and that the serine phosphorylation of the N-terminal domain does not play an essential role during epithelial cell differentiationThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author