Organization and structure of actin filament bundles in Listeria‐infected cells

Abstract

During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria-column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria-associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: 1) the surface of the bacteria, 2) the cytoplasm next to the bacteria, 3) the outer shell of the actin column, and 4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells.

Actin and alpha‐actinin dynamics in the adhesion and motility of EPEC and EHEC on host cells

“…This is consistent with transmission electron micrographs that do not reveal any signs of long parallel actin filaments in pedestals [Knutton et al, 1987;Kaper et al, 2004]. Steady state fluorescence polarization methods that predicted aligned filaments around the periphery of Listeria actin tails [Zhukarev et al, 1995] failed to detect aligned actin filaments in EPEC pedestals (Zhukarev et al, unpublished observations).…”

VASP protects actin filaments from gelsolin: An in vitro study with implications for platelet actin reorganizations

“…Failure to detect VASP within the actin tail using immunofluorescence could be because the actin filaments in the tail are too tightly packed to allow the antibody to bind. Indeed, it was initially even difficult to label them with the S1 fragment of myosin, a 110 kD protein smaller than antibodies [Tilney et al, 1992;Zhukarev et al, 1995]. Alternatively, VASP may not serve to stabilize the Listeria-induced actin filaments in the tail, since alpha-actinin also found there could serve this role in this system [Dold et al, 1994;Nanavati et al, 1994].…”

Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails

“…Several studies using light microscopy and EM have attempted to visualize the supramolecular organization of Listeria cytoplasmic comet tails and protrusions (1)(2)(3)(4)(5)(6)(7)(8). Despite advances in superresolution fluorescence microscopy, this technique has not yet resolved individual actin filaments in crowded environments.…”

“…Furthermore, conventional EM applied to detergent-extracted and dehydrated samples suffers from artifacts or a complete collapse of the delicate cytoskeletal networks. Cytoplasmic comet tails were reported to consist of multiple short actin filaments forming a cross-linked and branched network (1, 2) with some degree of alignment at their periphery (7). Branching occurs at the bacterial surface through the interaction of the bacterial surface protein ActA with the Arp2/3 complex (9, 10), which nucleates daughter filaments at an angle of 70°from preexisting filaments (10)(11)(12).…”