Joliot & Vriz

Homeoproteins and Cell Plasticity

Principal Investigators: Alain JOLIOT, DR2 Cnrs & Sophie VRIZ Pr. Paris Diderot

Redox Regulation of protein trafficking in Plasticity and Morphogenesis

Oxidative stress, which results from the imbalance in redox homestasis, is a toxic process that irreversibly modifies proteins, nucleic acids and lipids. Although it can result from external cues, it also find its origin from the production by cells of Reactive oxygen species (ROS), as by-products of aerobic cellular energy metabolism. Elevated ROS production has long been exclusively associated with pathological situations, including cancer, neurodegenerative diseases or diabetes. Recent findings now show that ROS also contribute to bona fide physiological processes, leading to a new paradigm known as redox signalling. Analogous to phosphorylation, Redox signalling consists in reversible protein post-translational modifications that mostly targets cysteine and to lower extent, methionine, two amino acids which oxidation state is known to regulate and fine-tune protein function in physiological situations. With the discovery of cellular pathways relying on “cysteine switches”, our perception of ROS has thus shifted from purely toxic compounds to bona fide second messengers. The main cellular sources of ROS reside in the mitochondria and at the plasma membrane, and both are subject to important crosstalk. Although the relevance of redox signalling in physiological and pathological contexts now becomes widely accepted, the identification of the downstream effectors and of the cellular processes targeted remains to be determined.

Visualising ROS
ROS levels results from a complex combination of enzymatic and non-enzymatic reactions, for its production and its degradation. Visualising ROS is thus critical to analyse its function. We recently developed a transgenic fish and stable HeLa cell lines expressing Hyper, a ratiometric fluorescent sensor for hydrogen peroxide (H2O2), one of the most relevant ROS endowed with signaling properties. We showed that H2O2 levels are highly dynamic both in time and space during embryonic development and adult regeneration (Figure 1). In HeLa cells, they show a highly specific subcellular distribution (Figure 1).

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Figure 1: Sensing H2O2. HyPer H2O2 ratiometric probe reveals spatial and temporal dynamic of H2O2 levels during zebrafish development, adult caudal fin regeneration and significant subcellular heterogeneities in HeLa cells.

ROS action
We discovered that the maintenance of high H2O2 levels for several hours at the plane of amputation is required to induce regenerative programs in vertebrates and stimulate cellular plasticity. We next have extended the demonstration of ROS action to other physiological situations, in adult mammals and during embryonic development (Figure 2), and identified nerves as critical players in redox signalling. High H2O2 level environment promotes cell plasticity and pro-regenerative processes, such as progenitor recruitment and blastema formation.

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Figure 2: Interplay between nerves and H2O2 during the regeneration (upper panel) and development (lower panel).
Regeneration. Immediately after amputation (dashed line), H2O2 (blue) start to accumulate in the stump epidermis. Few hours after amputation, the Wallerian degeneration begins and the nerves (red lines) recede along the stump while H2O2 levels further increase at the tip. The newly established H2O2 gradient then attracts the nerves, which regrow toward the tip of the stump, until they re-innervate the entire appendage. Nerve arrival switches off H2O2 production, which is no longer detected, and allows the regeneration of the missing part of the paw to proceed.
Development. The developing tissues/organs of the embryo (white circle) exhibit some areas with higher concentrations of H2O2 (blue area), which acts as an attractant for nerves (red lines). As nerves populate their target area, the concentration of H2O2 gradually decreases down to levels observed in the surrounding tissues, concomitantly with the completion of the innervation process.

We are developing several strategies to manipulate H2O2 levels in order to reproduce in a controlled way the extreme dynamics of H2O2 levels observed in physiological situations. In addition, the targeting this strategy to specific subcellular compartments has allowed us to address the role of H2O2 in protein trafficking, including protein secretion processes, conventional (Sonic Hedgehog) or unconventional (Engrailed homeoprotein), ex vivo and now also in vivo.

The Sonic Hedgehog (Shh) protein is a morphogen essential for the initiation of regeneration in adults, in part thanks to its ability to modulate redox levels. We now show that in turn, Shh trafficking is modulated by redox signaling (Figure 3). To further characterize which step(s) of Shh trafficking is/are regulated, we have adapted the Rush strategy developed by F.Perez (Institut Curie, Paris) to synchronize Shh secretion and carried out targeted mutagenesis of the cysteine residues within Shh which are putative effectors for redox signaling.

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Figure 3: Nerves, H2O2 and Shh interplay during adult zebrafish fin regeneration.
Following amputation, the injured nerves induce the production of H2O2 through the activation of the Hedgehog (Shh) pathway. Specific inhibition of Shh pathway prevents H2O2 production and consequently, regeneration.

Although first identified as transcriptional regulators, homeoproteins are able to transfer between cells through unconventional secretion and uptake pathways (Figure 4). We have identified the role of the phospholipid PIP2 in homeoprotein secretion, as reported by others for FGF2 and HIV Tat proteins, and more recently extend this role to homeoprotein uptake. Preliminary results indicate that homeoprotein trafficking is also regulated by redox signaling, possibly through its effect on PIP2 levels. The role of redox signaling on the dual mode of action of homeoprotein, either transcriptional or through its transfer between cells, is currently under investigation.

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Figure 4: Homeoprotein transfer. Homeoproteins are internalised by cells when adden in the medium (here vizualised with a recombinant fluorescent homeoprotein) and accumulates at the cell surface following secretion, through an unconventional pathway requiring PIP2. Together, these two processes result in the transfer of the protein between cells, allowing its paracrine action that superimpose on their classical transcriptional activity.

Our team is also engaged in the development of new tools and methods in collaboration with Arnaud Gautier and Ludovic Jullien (ENS, Paris) to visualize protein trafficking and secretion in live cells using fluorogen activated proteins.

Selected Publications

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Li, C., Mourton, A., Plamont, M.-A., Rodrigues, V., Aujard, I., Volovitch, M., Le Saux, T., Perez, F., Vriz, S., Jullien, L., Joliot A. & Gautier A. (2018). Fluorogenic Probing of Membrane Protein Trafficking. Bioconjug. Chem. 29, 1823–1828. Imperiali B: F1000Prime Recommendation in F1000Prime, 21-06-2018.

- Rampon, C., Volovitch, M., Joliot, A., and Vriz, S. (2018). Hydrogen Peroxide and Redox Regulation of Developments. Antioxidants 7, 159.

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Roux, L.N., Petit, I., Domart, R., Concordet, J.-P., Qu, J., Zhou, H., Joliot, A., Ferrigno, O., and Aberdam, D. (2018). Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein. Stem Cells 36, 1421–1429.

- Labit E, Rabiller L, Rampon C, Guissard C1, André M, Barreau C, Cousin B, Carrière A, Eddine MA, Pipy B, Pénicaud L, Lorsignol A, Vriz S, Dromard C, Casteilla L. (2018). Opioids prevent regeneration in adult mammals through inhibition of ROS production. Sci Rep 8, 12170.

- Meda, F., Rampon, C., Dupont, E., Gauron, C., Mourton, A., Queguiner, I., Thauvin, M., Volovitch, M., Joliot, A. & Vriz, S. (2017b), Nerves, H2O2 and Shh: Three players in the game of regeneration. Semin. Cell Dev. Biol. 80, 65–73.

- Meda, F., Joliot, A. & Vriz, S. (2017), Nerves and hydrogen peroxide: how old enemies become new friends. Neural Regen Res 12, 568–569. 

- Chen, Y., Ma, J., Lu, W., Tian, M., Thauvin, M., Yuan, C., Volovitch, M., Wang, Q., Holst, J., Liu, M., Vriz, S., Ye, S., Wang, L. & Li, D. (2016) Heritable expansion of the genetic code in mouse and zebrafish. Cell Res.27(2): 294-297. Comments in "Science Editors choice" Science (2017) 355:36

- Gauron, C., Meda, F., Dupont, E., Albadri, S., Quenech’Du, N., Ipendey, E., Volovitch, M., Del Bene, F., Joliot, A., Rampon, C. & Vriz, S. (2016), Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development. Dev. Biol. pii: S0012-1606 (15)30311-0.

- Plamont, M.-A., Billon-Denis, E., Maurin, S., Gauron, C., Pimenta, F.M., Specht, C.G., Shi, J., Quérard, J., Pan, B., Rossignol, J., Morellet, N., Volovitch, M., Lescop, E., Chen, Y., Triller, A., Vriz, S., Le Saux, T., Jullien, L. & Gautier, A. (2016), Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proc. Natl. Acad. Sci. U.S.A. 113, 497–502.

- Illien, F., Rodriguez, N., Amoura, M., Joliot, A., Pallerla, M., Cribier, S., Burlina, F., and Sagan, S. (2016). Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification. Sci Rep 6, 36938.

- Meda, F., Gauron, C., Rampon, C., Teillon, J., Volovitch, M. & Vriz, S. (2016), Nerves Control Redox Levels in Mature Tissues Through Schwann Cells and Hedgehog Signaling. Antioxid. Redox Signal. 24, 299–311.

- Dupont E., Prochiant, A. & Joliot A. (2015), Penetratin Story: An Overview. Methods Mol. Biol. 1324, 29–37.

- Rampon C., Gauron C., Lin T., Meda F., Dupont E., Cosson A., Ipendey E., Frerot A., Aujard I., Le Saux T., Bensimon D., Jullien L., Volovitch M., Vriz S. & Joliot, A. (2015), Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain. Development 142, 1840–1849.

- Gautier A., Gauron C., Volovitch M., Bensimon D., Jullien L. & Vriz S. (2014), How to control proteins with light in living systems. Nat. Chem. Biol. 10, 533–541.

- Rampon C., Gauron C., Meda F., Volovitch M. & Vriz S. (2014), Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration, Purinergic Signal, Aug 2.

- Vriz S., Reiter S. & Galliot B. (2014), Apoptosis : a program to regenerate. Curr Topics in Dev Biol. 108, 121–151.

- Gauron C., Rampon C., Bouzaffour M., Ipendey E., Teillon J., Volovitch M. & Vriz S. (2013), Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed. Scientific Reports 3, 2084.

- Feng Z., Zhang W., Xu L, Gauron C., Vriz S., Volovitch M., Jullien L., Weiss S. & Bensimon D. (2013), Optical control and study of biological processes at the single cell level in a live organism. Reports on Progress in Physics. 76(7): 072601.

- Fournier L., Gauron C., Xu L., Aujard I., Le Saux T., Gagey-Eilstein N., Maurin S., Dubruille S., Baudin J.-B., Bensimon D., Volovitch M., Vriz S. & Jullien L. (2013), A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation. ACS Chem. Biol. May7.

- Spatazza J., Lee H.H., Di Nardo A.A., Tibaldi L., Joliot A., Hensch T.K. & Prochiantz A. (2013), Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity. Cell Report, Volume 3, Issue 6,1815-1823.

- Spatazza J., Di Lullo E., Joliot A., Dupont E., Moya K.L. & Prochiantz A. (2013), Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed. Pharmacol Rev. Jan 8;65(1):90-104.

- Sagan S., Burlina F., Alves I.D., Bechara C., Dupont E. & Joliot A. (2013), Homeoproteins and homeoprotein-derived peptides: going in and out. Curr Pharm Des. 19(16):2851-62.

- Carlier, L., Balayssac, S., Cantrelle, F.-X., Khemtémourian, L., Chassaing, G., Joliot, A., and Lequin, O. (2013). Investigation of homeodomain membrane translocation properties: insights from the structure determination of engrailed-2 homeodomain in aqueous and membrane-mimetic environments. Biophys. J. 105, 667–678.

- Xu L., Feng Z., Sinha D., Ebenstein Y., Gauron C., Le Saux T., Lin S., Weiss S., Vriz S., Jullien L. & Bensimon D. (2012), Spatio-temporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization. Development, 139(18):3355-62. Eisen J: F1000Prime Recommendation In F1000Prime, 13 Sep 2012.

- Layalle S., Volovitch M., Mugat B., Bonneaud N., Parmentier M.-L., Prochiantz A., Joliot* A. & Maschat F.* (2011), Engrailed homeoprotein acts as a signaling molecule in the developing fly. Development, 138, 2315-2323.

People

Group leaders:
Joliot Alain, DR2 CNRS
Vriz Sophie, Professor Paris-Diderot

Senior researchers:
Rampon Christine, MDC Paris-Diderot

Postdoctoral fellows & PhD Students:
Mourton Aurélien, PhD student
Amblard Irene, PhD student
Thauvin Marion, PhD student

Technical staff:
Dupont Edmond, IR1 CNRS
Queguiner Isabelle, AI CDF
Lebled Valérie, TCN CNRS