Group leader: Alain Joliot

The main focus of this team is the analysis of the movement properties of homeoproteins at the cellular and molecular level.

I. Research Interests:

I.1 Background

In addition to their well known properties of transcriptional regulators, some homeoproteins have unexpected behaviour in cell culture systems. They shuttle continuously between nucleus and cytoplasm and they are able to move from cell to cell, without any degradation. This latter process imply both secretion and internalisation steps, which should rely on unconventional mechanisms. First, even homeoproteins are devoid of classical secretion signal, a pool of the protein accumulates in vesicular compartments, with a preferential association for cholesterol,GSL enriched membranes (rafts, microdomains, caveolae). Secondly, homeoproteins are internalised both at 37�C and 4�C, ruling out classical endocytosis. Once internalised homeoproteins accumulates preferentially in the nucleus, and are still able to interact with their DNA targets.

At the molecular level, all the sequences required for homeoprotein movements (both intra- and extra-cellular) reside within the homeodomain. The third alpha helix is necessary and sufficient for internalisation and an adjacent sequence (just upstream the third helix), is required both for secretion and nuclear export.

I.2 Recent work

Regulation of homeoprotein intercellular transfer

Engrailed homeoprotein intercellular transfer is tightly regulated by the phosphorylation state of the protein. Through the phosphorylation of a serine rich domain (outside the homeodomain) by protein kinase CK2, the transfer of the protein is dramatically inhibited at the level of its secretion. Differential accessibility of these target serine residues to CK2 (a constitutively active kinase) through protein conformation or protein-protein interactions, seems to be a major determinant of the phosphorylation state of the protein.

Trancytosis vectors

A synthetic peptide containing both "secretion" and "internalisation" signals derived from the homeodomain efficiently transfer through epithelial monolayer (MDCK). This transfer required the presence of both signals and is highly polarised from baso-lateral to apical. As opposed to internalisation, peptide secretion in the apical compartment is inhibited at 4�C. Secretion of the peptide correlates with its association with vesicular compartments, although Brefeldin A has no effect on this process.

I.3 Projects

Mechanism of secretion

Thanks to the identification of the sequence responsible for their unconventional secretion, homeoproteins represent an interesting paradigm for the study of this process, which has been observed for a variety of proteins. Synthetic peptides and homeoprotein mutants will be employed for the characterisation of the cellular and the molecular components involved in the secretion. This project will gather different approaches including, immunocytochemistry, sub-cellular and affinity chromatography. This study will be further extended to other class of proteins secreted by unconventional mechanisms in order to address the question of a conservation of these mechanisms.

Regulation of secretion

Regulation of Engrailed intercellular transfer through phosphorylation by CK2 is specific to this class of homeoproteins (it involves non-homeodomain sequences). We speculate that others regulatory events should occur for other class of homeoproteins. This hypothesis will be tested by the comparison of the movement properties of different classes of homeoproteins and their cognate homeodomains (the "driving" domain). The physiological significance of Engrailed intercellular transfer will be address by the analysis of Engrailed phosphorylation during development. A thigh regulation of this post-translational modification could have fundamental consequences at the physiological level as it could restrict intercellular transfer both spatially and temporally to specific situations during embryogenesis

II. Publications

ARTICLES

A. Maizel, O. Bensaude, A. Prochiantz and A. Joliot. A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development 126, 3183-90 (1999).

A. Joliot, A. Maizel, D. Rosenberg, A. Trembleau, S. Dupas, M. Volovitch and A. Prochiantz. Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr Biol 8, 856-63 (1998).

A. Joliot, A. Trembleau, G. Raposo, S. Calvet, M. Volovitch and A. Prochiantz. Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties. Development 124, 1865-75 (1997).

M. P. Schutze-Redelmeier, H. Gournier, F. Garcia-Pons, M. Moussa, A. H. Joliot, M. Volovitch, A. Prochiantz and F. A. Lemonnier. Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo. J Immunol 157, 650-5 (1996).

L. Chatelin, M. Volovitch, A. H. Joliot, F. Perez and A. Prochiantz. Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech Dev 55, 111-7 (1996).

L. Crews, P. B. Gates, R. Brown, A. Joliot, C. Foley, J. P. Brockes and A. A. Gann. Expression and activity of the newt Msx-1 gene in relation to limb regeneration. Proc R Soc Lond B Biol Sci 259, 161-71 (1995).

D. Derossi, A. H. Joliot, G. Chassaing and A. Prochiantz. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 269, 10444-50 (1994).

E. Bloch-Gallego, I. Le Roux, A. H. Joliot, M. Volovitch, C. E. Henderson and A. Prochiantz. Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro. J Cell Biol 120, 485-92 (1993).

I. Le Roux, A. H. Joliot, E. Bloch-Gallego, A. Prochiantz and M. Volovitch. Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties. Proc Natl Acad Sci U S A 90, 9120-4 (1993).

F. Perez, A. Joliot, E. Bloch-Gallego, A. Zahraoui, A. Triller and A. Prochiantz. Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide. J Cell Sci 102, 717-22 (1992).

A. H. Joliot, A. Triller, M. Volovitch, C. Pernelle and A. Prochiantz. alpha-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol 3, 1121-34 (1991).

M. R. Hirsch, I. Valarche, H. Deagostini-Bazin, C. Pernelle, A. Joliot and C. Goridis. An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains. FEBS Lett 287, 197-202 (1991).

A. Joliot, C. Pernelle, H. Deagostini-Bazin and A. Prochiantz. Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci U S A 88, 1864-8 (1991).

REVIEWS

E. Dupont, A. Joliot and A. Prochiantz (2002) Penetratins. in : CRC Handbook on Cell Penetrating Peptides, æ.Langel, Editor, CRC Press, in press.

A. Joliot, D. Derossi, S. Calvet and A. Prochiantz. Homeodomain and Homeodomain-Derived Peptides: New Vectors for Internalization of Molecules into Living Cells,Cell Biology: a laboratory handbook, Second Edition 4 111-119 (Academic Press, 1998).

A. Joliot, I. Le Roux, M. Volovitch, E. Bloch-Gallego and A. Prochiantz. Neurotrophic activity of a homeobox peptide. Prog Neurobiol 42, 309-11 (1994).

A. Joliot, I. Le Roux, M. Volovitch, E. Bloch-Gallego and A. Prochiantz. Neurotrophic activity of an homeobox peptide. Ann Genet 36, 70-2 (1993).

A. Joliot, I. Le Roux, M. Volovitch, E. Bloch-Gallego and A. Prochiantz. [Neurotrophic activity of homeopeptide]. C R Seances Soc Biol Fil 187, 24-7 (1993).

M. Volovitch, I. le Roux, A. H. Joliot, E. Bloch-Gallego and A. Prochiantz. Control of neuronal morphogenesis by homeoproteins: consequences for the making of neuronal networks. Perspect Dev Neurobiol 1, 133-8 (1993).

A. Prochiantz, A. H. Joliot, M. Volovitch and A. Triller. Have homeoproteins autocrine and paracrine activities? Implications for our understanding of cellular recognition during development. Comments Dev. Neurobiol. 1, 359-372 (1992).

A. Joliot, A. Triller, M. Volovitch and A. Prochiantz. [Are embryonic forms of NCAM homeobox receptors?]. C R Acad Sci III 314, 59-63 (1992).

PATENTS

A. Prochiantz et A. Joliot. Nouveaux facteurs de croissance comprenant un peptide hom¹obo”te. Juin 1990) N�90 06912. ( Extension aux pays Europ¹ens, Suisse, Japon, USA et Cor¹e du Sud).

E. Dupont, A. Joliot and A. Prochiantz. Vecteurs de transport ù travers un ¹pith¹lium ù jonctions serr¹es. 20 Novembre 2000, N� 0014945

III. Members of the group:

Alain Joliot: joliot@wotan.ens.fr

Edmond Dupont :Post-doc

Val¹rie Lebled: technician

Michel Tassetto: PhD student

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