Motility of ActA protein-coated microspheres driven by actin polymerization

Abstract

Actin polymerization is required for the generation of motile force at the leading edge of both lamellipodia and filopodia and also at the surface of motile intracellular bacterial pathogens such as Listeria monocytogenes. Local catalysis of actin filament polymerization is accomplished in L. monocytogenes by the bacterial protein ActA. Polystyrene beads coated with purified ActA protein can undergo directional movement in an actin-rich cytoplasmic extract. Thus, the actin polymerization-based motility generated by ActA can be used to move nonbiological cargo, as has been demonstrated for classical motor molecules such as kinesin and myosin. Initiation of unidirectional movement of a symmetrically coated particle is a function of bead size and surface protein density. Small beads (<0.5 m in diameter) initiate actin-based motility when local asymmetries are built up by random f luctuations of actin filament density or by thermal motion, demonstrating the inherent ability of the dynamic actin cytoskeleton to spontaneously self-organize into a polar structure capable of generating unidirectional force. Larger beads (up to 2 m in diameter) can initiate movement only if surface asymmetry is introduced by coating the beads on one hemisphere. This explains why the relatively large L. monocytogenes requires polar distribution of ActA on its surface to move.Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that moves rapidly through host cells or cytoplasmic extracts, propelled by actin hijacked from the host cell (1-3). Moving bacteria are associated with an actin-rich structure that resembles the tail of a comet (4). The actin filaments within the comet tail remain fixed in space as the bacterium moves, and the rate of bacterial movement is tightly correlated with the rate of actin filament polymerization at the front of the comet tail (5, 6). Because no motor proteins have been found to be involved in this form of motility, filament elongation is thought to provide the force necessary to propel the bacterium through the cytoplasm (7). The comet tail structure is functionally similar to the actin meshwork in the lamellipodia of a locomoting eukaryotic cell, and the surface of the bacterium is analogous to the leading edge. Therefore, insights into the mechanism of actin-based bacterial movement should help explain the mechanisms of eukaryotic actin-based motility.A bacterial comet tail is made up of short (0.2-0.3 m) actin filaments crosslinked together to form a dense cylindrical structure (4). Other intracytoplasmic pathogens form similar comet tail structures and undergo actin-based motility, including the unrelated bacteria Shigella flexneri (8) and Rickettsia spp. (9) and vaccina virus (10). In uninfected cells and cytoplasmic extracts, actin-rich comet tails are associated with moving endosomes (J. Heuser, personal communication) and phospholipid vesicles (11). Although much is understood about the biochemical mechanisms of catalyzing actin filament polymerization in several of th...

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…In addition, ActAcoated latex beads are able to polymerize actin in cellular extracts in vitro. 162 To stimulate actin polymerization, ActA mimics the activity of the WASP family proteins, which are host cell actinnucleating factors. 163 The N-terminal region of ActA is sufficient for its activity as it contains consensus sequences present in proteins of the WASP family, including an actin monomer-binding domain and two acidic regions that interact with and activate the Arp2/3 complex, a host actin nucleator.…”

Decoupling the Coupling: Surface Attachment in Actin-Based Motility

“…Bacterial motility can be reconstituted in an in vitro system where the cytosol is replaced by a defined mixture of purified components (7). Furthermore, artificial objects such as plastic beads (8) or lipid vesicles (9,10) coated with the bacterial protein ActA or with another NPF such as N-WASP (11) are also able to form actin comet tails and move in a remarkably lifelike manner in cytoplasmic extracts or reconstituted systems. ABSTRACT Actin filament polymerization provides the driving force for several kinds of actinbased motility, propelling loads such as the plasma membrane at the leading edge of a crawling cell, an endosomal vesicle, or an intracellular bacterial pathogen.…”

Nucleation geometry governs ordered actin networks structures

“…The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] . However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied 14,19 .…”