Accumulation of profilin II at the surface of Listeria is concomitant withthe onset of motility and correlates with bacterial speed

The spatial and temporal activity of the actin cytoskeleton is precisely re gulated during cell motility by several microfilament-associated proteins o f which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Lis teria-infected cells. Among the different GFP-profilin fusion proteins stud ied, only the construct in which the GFP moiety was fused to the carboxy te rminus of profilin II (profilin II-GFP) was recruited by intracellular List eria, The in vitro ligand-binding properties of this construct, e.g. the bi nding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisph osphate (PIP2), were unaffected by GFP, Profilin II-GFP co-localised with v inculin and Mena to the focal adhesions in REF-52 fibroblasts and was distr ibuted as a thin line at the front of protruding lamellipodia in B16-F1 mou se melanoma cells. In Listeria-infected cells, profilin II-GFP was recruite d, in an asymmetric fashion, to the surface of Listeria at the onset of mot ility whereas it was not detectable on nonmotile bacteria. In contrast to t he vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescenc e intensity of profilin II-GFP directly correlated with the speed of the ba cteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.