Review
Listeria comet tails: the actin-based motility machinery at work

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Listeria monocytogenes is a master of mimicry that uses the host cell actin system both to move within the cytoplasm of infected cells and for cell-to-cell spread. Recent studies of Listeria and similarly acting pathogens have generated leaps in our understanding of the actin-based force producing machinery. This machinery is essential for most motile properties of cells, not least for cell migration. In a minimal configuration, it consists of the Arp2/3-complex, Ena–VASP proteins, cofilin, capping protein and a nucleation-promoting factor. In this review, we discuss current models of pseudopodial protrusions and describe how the road to more complex models lies open and is already paved by recent studies using Listeria-based biomimetic motility assays.

Introduction

Cellular motility encompasses processes that enable cells to change shape, divide, extend membrane protrusions and migrate; it also includes the transport of vesicles within the cytoplasm. Cell motility consequently constitutes a crucial aspect of eukaryotic development and survival and is central in several pathologies. Most of these motile features rely on dynamic reorganizations of the filamentous actin system, a major component of the cell skeleton [1]. Understanding at a molecular level how the dynamic assembly of actin filaments is temporally and spatially regulated by the multiple actin-modulating proteins that are simultaneously expressed in cells – and how this supports cellular motility – continues to be a major research focus. Several proteins are known to regulate actin filament nucleation, polymerization and stability. Figure 1a–e provides an introduction to actin filament dynamics in the context of lamellipodia and filopodia formation.

This review describes how the study of the facultative intracellular pathogen Listeria monocytogenes has provided fundamental breakthroughs in understanding actin-based propulsion, and how this species continues to form an important model to address new aspects in the field of cell motility. L. monocytogenes is a ubiquitous Gram-positive pathogen that causes listeriosis, a disease characterized by severe gastroenteritis, encephalitis, meningitis or septicemia, as well as miscarriage. Listeria is capable of infecting its host by actively invading non-phagocytotic cells. In these host cells, the bacteria characteristically use the actin-based motility machinery to move intracellularly (http://cmgm.stanford.edu/theriot/movies.htm) and to spread to adjacent cells. In this way, they stay largely protected from circulating immune cells (see Box 1 and references therein). By true subversive action (i.e. by mimicking and dominating over host cell actin filament nucleation-promoting factors [NPFs]), the bacterial ActA protein (Figure 1f) suffices to recruit the host actin-binding proteins needed to assemble the actin-based force-producing machinery [2]. This manifests itself by the characteristic comet tails, which are composed of growing actin filaments and which push the bacterium through the cytosol at high speed. Since the discovery of this property, studies have identified several unrelated pathogens that have developed similar strategies to manipulate the cytoskeletal machinery of the host cell for movement through the cytoplasm and for cell-to-cell spread [3]. These strategies analogously combine structural and functional mimicry of host proteins and are described in Box 2.

Study of Listeria and similarly acting pathogens has provided exciting shortcuts in the discovery and/or functional definition of key modulators of actin nucleation in cells; for example, the complex between the actin-related proteins 2 and 3 (Arp2/3-complex), Enabled–Vasodilator-stimulated phosphoproteins (Ena–VASPs) and the family of Wiskott Aldrich Syndrome Proteins–(WASPs) and WASP verprolin (Wave) homology proteins and their activators. Here, we focus on the molecular mechanisms of Listeria bacterial intracellular motility that contribute to our understanding of how cellular actin filament nucleation and elongation events act in extending membrane protrusions. We comment on the usefulness of cataloguing the components of Listeria comet tails and describe how Listeria inspired the development of biomimetic motility tools consisting of beads or vesicles coated with NPFs or ActA. These tools are increasingly being used to dissect effects of novel modulators of actin-based motility that are active in different types of cellular protrusions. These biomimetics also paved the way to a better biophysical understanding of the propulsive force generated by actin polymerization, a necessary aspect to fully grasp cell protrusion. A comprehensive description of the biophysical aspects of force generation is beyond the scope of this review; seminal updates are provided elsewhere 4, 5, 6.

Section snippets

Listeria actin filament nucleation-promoting factor ActA: master of mimicry

The characteristic actin comet tail at one pole of a bacterium enables it to cruise through the cytosol. This tail is formed by actin polymerization, which is initiated by a protein complex containing the asymmetrically distributed bacterial surface protein ActA and several host factors such as VASP and the Arp2/3-complex [7]. ActA is necessary and sufficient for bacterial motility and is crucial for pathogenesis in the murine model of infection. When expressed in the non-motile Listeria innocua

Listeria propulsion and models of Arp2/3-dependent cellular protrusion

The most fascinating accomplishment of studies on intracellular pathogenic bacteria is the elucidation of cellular actin filament-nucleating machineries. Identifying both the key role of the Arp2/3-complex in bacterial intracellular motion and the minimal machinery have indeed provided fertile grounds for the current model of lamellipodial outgrowth: the dendritic nucleation and array-treadmilling model (for a review see [28]). In this model, the lamellipodial network, which constitutes the 2

Biomimetic assays yield growing insight into cellular protrusions: the Listeria legacy

Listeria or Listeria-derived biomimetic motility assays using beads or vesicles coated with ActA or NPFs (e.g. the VCA domain of WASP) are increasingly being used to reveal the possible role of proteins in lamellipodial actin dynamics. These test systems consequently constitute an important bridging function, linking biochemistry to cell biological interpretation. This approach, for example, supplied insight into the relative role of the Arp2/3-complex and VASP in propulsive movement [43].

Addressing Listeria comet tail complexity as the basis to extend current models of cell protrusion

A growing portfolio of cell biological techniques (e.g. quantitative fluorescent speckle microscopy, fluorescence resonance energy transfer [FRET], fluorescence recovery after photobleaching [FRAP], fluorescence loss in photobleaching [FLIP]) and cell-free systems and tools, such as the biomimetics described above, are available to researchers in the motility field. This will increasingly enable workers to address – both experimentally and computationally – the higher complexity of cellular

Conclusions

Studying the intricate strategies used by L. monocytogenes and other pathogens to move intracellularly has been to the migration field what ‘that admired teacher at high school’ has been to many of us: highly instructive and inspiring. Studying these bacteria has revealed many key molecular players of actin dynamics and continues to do so. In addition, researchers in turn – and with matched creativity – developed tools that mimic the Listeria strategy. These biomimetics have enabled workers to

Acknowledgements

A.L. is a postdoctoral researcher of the Fund for Scientific Research (FWO-Vlaanderen). P.C is an International Scholar of the Howard Hughes Medical Institute. This work received financial support from the Concerted Research Actions (GOA) of the Ghent University, Belgium and the Fund for Scientific Research (FWO-Vlaanderen), the Ministère de la Recherché et de la Technologie (MRT), Inserm and Institut Pasteur.

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