ACTIN-BASED MOVEMENT OF LISTERIA-MONOCYTOGENES - ACTIN ASSEMBLY RESULTS FROM THE LOCAL MAINTENANCE OF UNCAPPED FILAMENT BARBED ENDS AT THE BACTERIUM SURFACE

The thermodynamic basis for actin-based motility of Listeria monocytog enes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 mu M) is accounted for by the sequestering func tions of T beta(4)(Xen) (20 mu M) and profilin (5 mu M), the barbed en ds being capped. Movement of Listeria was not abolished by depletion o f over 99% of the endogenous profilin. The proline-rich sequences of A ctA are unlikely to be the target of profilin. All data support the vi ew that actin assembly at the rear of Listeria results from a local sh ift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i. e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin a t a high concentration. The role of membrane components is demonstrate d by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enric hed in membranes; (b) Actin-based motility of endogenous vesicles, exh ibiting the same rocketing movement as Listeria, can be observed in th e extracts.