Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.

Role of PI(4,5)P2 and Cholesterol in Unconventional Protein Secretion.

Besides its protective role in the maintenance of cell homeostasis, the plasma membrane is the site of exchanges between the cell interior and the extracellular medium. To circumvent the hydrophobic barrier formed by the acyl chains of the lipid bilayer, protein channels and transporters are key players in the exchange of small hydrophilic compounds such as ions or nutrients, but they hardly account for the transport of larger biological molecules. Exchange of proteins usually relies on membrane-fusion events between vesicles and the plasma membrane. In recent years, several alternative unconventional protein secretion (UPS) pathways across the plasma membrane have been characterised for a specific set of secreted substrates, some of them excluding any membrane-fusion events (Dimou and Nickel, Curr Biol 28:R406-R410, 2018). One of thesbe pathways, referred as type I UPS, relies on the direct translocation of the protein across the plasma membrane and not surprisingly, lipids are essential players in this process. In this chapter, we discuss the roles of phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and cholesterol in unconventional pathways involving Engrailed-2 homeoprotein and fibroblast growth factor 2.

Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.

Nerves, H2O2 and Shh: Three players in the game of regeneration.

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H2O2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H2O2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H2O2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H2O2 levels to their original state. A similar regulatory loop between H2O2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity.

The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo.

The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein.

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.

Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

Fluorogenic Probing of Membrane Protein Trafficking.

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.

Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed.

Homeoproteins constitute a major class of transcription factors active throughout development and in adulthood. Their membrane transduction properties were discovered over 20 years ago, opening an original field of research in the domain of vector peptides and signal transduction. In early development, homeoprotein transfer participates in tissue patterning, cell/axon guidance, and migration. In the axon guidance model, homeoproteins exert their non-cell autonomous activity through the regulation of translation, in particular, that of nuclear-transcribed mitochondrial mRNAs. An important aspect of these studies on patterning and migration is that homeoproteins sensitize the cells to the action of other growth factors, thus cooperating with established signaling pathways. The role of homeoprotein signaling at later developmental stages is also of interest. In particular, the transfer of homeoprotein Otx2 into parvalbumin-expressing inhibitory neurons (PV-cells) in the visual cortex regulates cortical plasticity. The molecular deciphering of the interaction of Otx2 with binding sites at the surface of PV-cells has allowed the development of a specific Otx2 antagonist that reopens plasticity in the adult cortex and cures mice from experimental amblyopia, a neurodevelopmental disease. Finally, the use of homeoproteins as therapeutic proteins in mouse models of glaucoma and Parkinson disease is reviewed. In the latter case, engrailed homeoproteins protect mesencephalic dopaminergic neurons by increasing the local translation of complex I mitochondrial mRNAs. In conclusion, this review synthesizes 20 years of work on the fundamental and potentially translational aspects of homeoprotein signaling.

Engrailed homeoprotein acts as a signaling molecule in the developing fly.

Homeodomain transcription factors classically exert their morphogenetic activities through the cell-autonomous regulation of developmental programs. In vertebrates, several homeoproteins have also been shown to have direct non-cell-autonomous activities in the developing nervous system. We present the first in vivo evidence for homeoprotein signaling in Drosophila. Focusing on wing development as a model, we first demonstrate that the homeoprotein Engrailed (En) is secreted. Using single-chain anti-En antibodies expressed under the control of a variety of promoters, we delineate the wing territories in which secreted En acts. We show that En is a short-range signaling molecule that participates in anterior crossvein development, interacting with the Dpp signaling pathway. This report thus suggests that direct signaling with homeoproteins is an evolutionarily conserved phenomenon that is not restricted to neural tissues and involves interactions with bona fide signal transduction pathways.

Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube.

Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. Here, we have used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. We report the extracellular expression of Pax6 and the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity.

Investigation of homeodomain membrane translocation properties: insights from the structure determination of engrailed-2 homeodomain in aqueous and membrane-mimetic environments.

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.

Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods.

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates.

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

Unconventional Secretion, Gate to Homeoprotein Intercellular Transfer.

Unconventional secretion allows for the secretion of fully mature and biologically active proteins mostly present in the cytoplasm or nucleus. Besides extra vesicle-driven secretion, non-extravesicular pathways also exist that specifically rely on the ability of the secreted proteins to translocate directly across the plasma membrane. This is the case for several homeoproteins, a family of over 300 transcription factors characterized by the structure of their DNA-binding homeodomain. The latter highly conserved homeodomain is necessary and sufficient for secretion, a process that requires PI(4,5)P2 binding, as is the case for FGF2 and HIV Tat unconventional secretion. An important feature of homeoproteins is their ability to cross membranes in both directions and thus to transfer between cells. This confers to homeoproteins their paracrine activity, an essential facet of their physiological functions.

Anionic lipids induce a fold-unfold transition in the membrane-translocating Engrailed homeodomain.

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.

Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway.

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

Homosalate boosts the release of tumour-derived extracellular vesicles with protection against anchorage-loss property.

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs' release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate-, but not by Bafilomycin A1-treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.

Transduction peptides: from technology to physiology.

During the past fifteen years, a variety of peptides have been characterized for their ability to translocate into live cells. Most are efficient vectors that can internalize hydrophilic cargoes, and so provide a valuable biological (and potentially therapeutic) tool for targeting proteins into cells. Furthermore, translocation of cell-permeable peptides across the plasma membrane and their subsequent access to the cytosol, even when fused to large hydrophilic proteins, is challenging the perception of the plasma membrane as an impermeable barrier.