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

Abstract

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

Profilin Isoforms in Health and Disease – All the Same but Different

“…The increasingly improving CRISPR/Casbased methods will open new avenues to study profilins by loss of function, gene-tagging, and gain of function experiments in a great variety of cell types and organisms (see for review (Pickar-Oliver and Gersbach, 2019). Particularly tagging of endogenous PFN1 will be feasible by new profilin constructs internally fused to fluorescent proteins (Nejedla et al, 2017), as they circumvent the impaired ligand binding of traditional GFP-fused PFN1 variants (Geese et al, 2000;Wittenmayer et al, 2000). A better insight into the functions of profilin and its isoforms will help us to understand fundamental cell biological mechanisms in health and disease.…”

A crucial role for profilin–actin in the intracellular motility of Listeria monocytogenes

“…The L. monocytogenes mutant ActA5 (Niebuhr et al ., 1997) and wild‐type strains were grown in brain–heart infusion broth (BHI; DIFCO Laboratories) as described previously (Geese et al ., 2002). PtK2 and MDCK cells were grown as described in Geese et al . (2000).…”

“…Thus, it is possible that the impairment of Listeria motility is due to the binding of PxA to the (+) ends of the actin filaments that abut the bacterial surface. As the targeting of profilin, and possibly profilin-actin complexes, to the bacterial surface is mediated by the interaction with the central proline-rich region of Ena/VASP proteins (Geese et al, 2000(Geese et al, , 2002, the localization of which at this site is not affected by PxA (see supplementary information), it is possible that this interaction is required for the inhibitory effect of PxA on bacterial motility.…”

“…Among the proteins that have been implicated in the regulation of actin‐filament dynamics is profilin, which was originally discovered as an actin‐monomer‐binding protein (Carlsson et al ., 1977). Profilin is localized to highly dynamic regions of cultured cells, such as the tips of spreading lamellipodia and focal adhesions (Theriot & Mitchison, 1993; Geese et al ., 2000), and also accumulates at the rear end of motile Listeria (Theriot et al ., 1994; Smith et al ., 1996; Geese et al ., 2000). The deletion of the genes encoding profilin alters the architecture of the actin cytoskeleton, severely impairs cell migration and leads, in most cases, to lethality (Balasubramanian et al ., 1994; Haugwitz et al ., 1994; Witke et al ., 2001; Schlüter et al ., 1997).…”

Actin Filament Barbed End Elongation with Nonmuscle MgATP−Actin and MgADP−Actin in the Presence of Profilin

“…Profilin desequesters actin monomers bound to thymosin β4, allowing actin filament barbed end elongation (6), and profilin enhances actin filament turnover in the presence of ADF/cofilin (7). Profilin recruits monomeric MgATP-actin to areas of actin filament growth via interactions with N-WASP (1,2) and VASP (8)(9)(10)(11). When the actin filament barbed ends are capped by other actin-binding proteins, profilin acts as an actin-sequestering protein.…”