Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc

Although motile endocytic vesicles form actin-rich rocket tails Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells.