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 homeostasis, is a toxic process that irreversibly modifies proteins, nucleic acids and lipids. Although it can result from external cues, it also finds 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.
ROS levels results from a complex combination of enzymatic and non-enzymatic reactions, for their (ROS) production and their degradation. Visualising ROS is thus critical to analyze their 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).
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.
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.
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 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.
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.
Figure 4: Homeoprotein transfer. Homeoproteins are internalised by cells when added 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.
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- 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.
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- 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.
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- 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.
Joliot Alain, DR2 CNRS
Vriz Sophie, Professor Paris-Diderot
Rampon Christine, MDC Paris-Diderot
Postdoctoral fellows & PhD Students:
Amblard Irene, PhD student
Thauvin Marion, PhD student
Dupont Edmond, IR1 CNRS
Queguiner Isabelle, AI CDF
Lebled Valérie, TCN CNRS