Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Thymosins in Health and Disease Multifunctionality of the β-thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing Clotilde Husson, 1 Franc ¸ois-Xavier Cantrelle, 2 Pierre Roblin, 1 Dominique Didry, 1 Kim Ho Diep Le, 1 Javier Perez, 3 Eric Guittet, 2 Carine Van Heijenoort, 2 Louis Renault, 1 and Marie-France Carlier 1 1,2 Centre de Recherche de Gif 1 LEBS-CNRS and 2 ICSN-CNRS, CNRS, Gif-sur-Yvette, France. 3 Synchrotron SOLEIL, Gif-sur-Yvette Cedex, France Addresses for correspondence: Marie-France Carlier and Louis Renault, Cytoskeleton Dynamics and Motility Group, UPR 3082, Carine van Heijenoort, ICSN, Laboratoire de chimie et biologie structurales, ICSN, UPR2301, France. Centre de Recherche de Gif, CNRS, 1 avenue de la Terrasse 91198 Gif-sur-Yvette, France. carlier@lebs.cnrs-gif.fr, renault@lebs.cnrs-gif.fr, and carine@icsn.cnrs-gif.fr The β-thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The β-thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F- actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first β-thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin–actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities. Keywords: actin; thymosin β4; WH2 domain; Spire; Cordon-Bleu Introduction Thymosin 4 (T4) is a functionally small multi- facetted heat-stable protein. Inside the cells it is a soluble cytoplasmic peptide that acts as the major sequesterer of G-actin. Hence its function is to cre- ate a reservoir of unassembled monomeric actin, which is used, upon de-sequestration, to promote much more extensive assembly of actin filaments than would be otherwise possible. It is present at very high concentrations (300–500 M) in platelets and lymphocytes, and overexpressed in several tu- mors. Thymosin 4 also acts extracellularly to en- hance embryonic endothelial cell migration, pro- mote dermal and corneal wound healing in a matrix metalloproteinase-dependent fashion, stim- ulate coronary vasculogenesis, thus preventing heart failure. 1 The latter activities of thymosin 4 make this peptide a potent therapeutic tool now in phase 1A and 1B trials for myocardial infarction. 2 The nature of the putative nonactin partners (recep- tors, signaling molecules) of thymosin 4 in its pleiotropic extracellular effects are not known, and actually some of its activities in tissue repair may be due to its being secreted in complex with actin, 3 whereas its tumor-suppressor activities may be due to its intracellular actin binding. 4 Understanding the multiple functions of thymosin 4 therefore re- quires extensive analysis of the structural basis for its versatile regulatory effects in actin assembly. Interest in this issue is increased by the fact that thymosin 4 appears as a model actin-binding peptide for doi: 10.1111/j.1749-6632.2010.05473.x 44 Ann. N.Y. Acad. Sci. 1194 (2010) 44–52 c  2010 New York Academy of Sciences.