Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy.

Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the µm²-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types. © 2018 by John Wiley & Sons, Inc.

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.

Annexin-A5 promotes membrane resealing in human trophoblasts.

Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout.

In most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells. We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE(cs)) by three base pairs. This non-palindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor alpha (hERalpha) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERalpha-ERE(cs) and hERalpha-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo. In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes.

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites.

BACKGROUND INFORMATION: Spherulites are multi-lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B-subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non-toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. RESULTS: We demonstrate that STxB-spherulites (ST x B-functionalized spherulites) are internalized into HeLa cells in a receptor-dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. CONCLUSIONS: Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB-spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB-spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.