In Brief

Listeria monocytogenes uses an actin tail to project itself into neighboring cells. This process allows the bacterium to cross the placental and blood-brain barriers and causes spontaneous abortion and meningitis, respectively, but it has only been documented in a few static electron micrographs. Now Robbins et al. (page 1333) observe intercellular Listeria movement in real time, and suggest that it may take advantage of a conserved host cell engulfment process.

Once a Listerium has formed an intercellular protrusion averaging 8.5 μm in length, the bacterium continues an erratic motility that Robbins et al. term “fitful movement.” This ceases after ∼20 min, presumably when the donor cell's membrane is sealed, cutting off the supply of ATP. Another ∼20 min later, the second (host cell) membrane fuses, and the protrusion shrinks to a roughly spherical double membrane vacuole. Within 5 min the vacuole acidifies and lyses to release the bacterium.

The forces driving the membrane fusions, and the reasons for the delay between fusion steps, are not known. Perhaps an actin-based structure must be built around the large neck of the protrusion. Although the process is not speedy on a bacterial time scale, the bacterium does appear to take advantage of host cell processes. Robbins et al. note that the actin tails in the protrusions are unusually stable, possibly because of actin-stabilizing proteins found near the membranes of microvilli. This may help the bacterium to launch itself into the projection. Clues regarding engulfment come from cells in uninfected epithelial cultures, which take up small quantities of membrane from their neighbors. Normally this may function in the exchange of membrane-bound ligands, and the phagocytosis of apoptotic cell fragments, but for Listeria this represents a perfect vehicle for cell-to-cell spreading.

Shigella flexneri is another bacterium that moves by coopting host cell actin. For both Listeria and Shigella, only one bacterial protein is needed to induce motility. The relevant Shigella protein, IcsA, binds to N-WASP, a protein that is activated by Cdc42 during the formation of filopodia. Now Egile et al. (page 1319) show that IcsA activates multiple functions of N-WASP that may work together to produce motility.

The NH₂ terminus of N-WASP binds to the side of actin filaments. The COOH-terminal VCA region of N-WASP (so named for its verprolin and cofilin homologies and an acidic segment) has two activities. The first is activation of actin nucleation by Arp2/3, an effect that is greatly enhanced by IcsA. IcsA appears to act like Cdc42 by exposing the region of VCA that interacts with Arp2/3 (and with monomeric actin; see below).

The second, and more surprising activity of the VCA region is profilin-like. VCA binds monomeric ATP-G-actin and delivers it to the barbed end of the actin filament (the end nearest the bacterium). By attaching actin filaments to the bacterial surface (via IcsA and F-actin binding) and feeding cyclic barbed end growth, N-WASP may use actin ATPase activity to act as an insertional motor. This idea of a step motor differs from the current Brownian ratchet model of actin-based motility, which relies on the flexibility of filaments for force production. Further experiments will be needed to distinguish between the two models.

Organelles must be partitioned equally during mitosis. Rogers et al. (page 1265) uncover one strategy by which pigment-filled melanosomes from frog melanophores reach an even distribution suitable for partitioning between daughter cells: the release of their myosin motor.

During interphase, myosin-based motility on randomly arranged actin filaments helps to disperse the melanosomes. But in mitosis much of the actin redistributes to the contractile furrow, whereas melanosomes must remain dispersed. Rogers et al. first confirmed that myosin V is the motor that drives actin-based melanosome motility, and then found that the motor is released by the addition of mitotic frog extract.

This may be the only option for turning off myosin V motility. The motor activity of some non-muscle myosins is regulated by phosphorylation at a serine in the motor domain, but in most myosins (including myosin V) this residue is a glutamate or aspartate. These residues are believed to result in a motor that is constitutively on, and therefore, the transport can be turned off only by releasing the motor from its cargo.

Mutation of the adenomatous polyposis coli (APC) gene initiates most human colorectal cancers, and APC is an essential inhibitor of the Wnt signaling pathway in mammals. Not surprisingly then, mutations in APC in mice are lethal. Yet in Drosophila, the only strong effects of dAPC mutations are in the developing eye. McCartney et al. (page 1303) and Yu et al. (Yu, X., L. Waltzer, and M. Bienz. 1999. Nat. Cell Biol. 1:144–151) resolve this discrepancy with the discovery of a second fly APC gene. Their analysis of the gene product suggests that its function involves interactions with the actin and possibly microtubule cytoskeletons, and with adherens junctions.

McCartney et al. used sequence database searching to identify a gene that they name dAPC2. The protein product is often found in association with either actin or microtubules, including spindles in cellularized embryos and a crescent located at one end of neuroblasts that are poised to undergo asymmetric division. “The localization of this protein is consistent with the idea that it might be part of the machinery that transduces a Wnt-like signal to the cytoskeleton,” says Mark Peifer.

The researchers isolated a temperature-sensitive dAPC2 mutant that appears to be a loss of function. In the embryo, the mutant has the same segment-polarity phenotype caused by ubiquitous expression of Wingless (Wg; the Drosophila Wnt homologue), and double mutant analysis further links dAPC2 to the Wg signaling pathway. So far, no aberrant phenotype is obvious in adult flies when the temperature is raised later in development. Coauthor Amy Bejsovec plans to look for more subtle defects and to test other dAPC2 mutants and double mutants with dAPC.

Wg and dAPC are known to converge on Armadillo, the Drosophila version of β-catenin. Arm is present predominantly in the zonula adherens, but Wg signaling leads to a cessation of Arm degradation by a complex including APC, Axin, and the kinase, Shaggy. The resulting pool of cytoplasmic Arm pairs with transcription factors and turns on proliferative genes.

Yu et al. isolated dAPC2 (which they named E-APC) by a two hybrid assay with Arm. They provide a detailed description of the epithelial localization also observed by McCartney et al. The E-APC2 is in the zonula adherens, in a pattern that overlaps Arm staining but is more punctate. Yu et al. postulate that this localization is necessary for E-APC2 function. Disruption of the zonula adherens, mutation of shaggy, or RNA interference with E-APC2 all have similar effects: E-APC2 delocalization and an increase in cytoplasmic Arm.

The phosphorylation of Arm that targets it for destruction appears to be handled by Shaggy and Axin. Senior author Mariann Bienz suggests that E-APC's role may be to regulate Arm destruction spatially, by gathering together Shaggy, Axin, and cytoplasmic Arm at the zonula adherens. This destruction complex would be poised to mop up any excess Arm released when epithelial cells are remodeled, and ideally placed to receive signals from nearby membrane receptors.