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The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Sechi AS, Wehland J, Small JV - J. Cell Biol. (1997)

Bottom Line: The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin.We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane.A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg. ase@gbf-brauschweig.de

ABSTRACT
Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

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Isolated pseudopodium (a, overview) labeled with smooth muscle myosin S-1 and showing, in b and c, the actin filament polarity at the two ends: (b) head end and (c) trailing end. The positions of the regions enlarged in b and c are indicated in a (white arrowheads). The loss of bacterium from the head end is a typical consequence of S-1 labeling. Bars: (a) 1 μm; (b and c) 0.2 μm.
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Figure 7: Isolated pseudopodium (a, overview) labeled with smooth muscle myosin S-1 and showing, in b and c, the actin filament polarity at the two ends: (b) head end and (c) trailing end. The positions of the regions enlarged in b and c are indicated in a (white arrowheads). The loss of bacterium from the head end is a typical consequence of S-1 labeling. Bars: (a) 1 μm; (b and c) 0.2 μm.

Mentions: To gain information about filament polarity, we also labeled isolated and Triton-extracted pseudopodia with myosin S-1, before fixation and negative staining. Since the labeling density was so high, it was difficult to ascertain arrowhead directions in the bulk of the tail. However, at the end of the tail, almost all filaments were oriented with their pointed ends towards the tip (Fig. 7). Interestingly, S-1 decoration commonly dislodged the bacterium from the head of the tail and revealed filaments, with few exceptions, showing barbed ends, in agreement with the data of Tilney et al. (1992a). Thus, we could confirm that the fast growing ends of the actin filaments abut the bacterium.


The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Sechi AS, Wehland J, Small JV - J. Cell Biol. (1997)

Isolated pseudopodium (a, overview) labeled with smooth muscle myosin S-1 and showing, in b and c, the actin filament polarity at the two ends: (b) head end and (c) trailing end. The positions of the regions enlarged in b and c are indicated in a (white arrowheads). The loss of bacterium from the head end is a typical consequence of S-1 labeling. Bars: (a) 1 μm; (b and c) 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139863&req=5

Figure 7: Isolated pseudopodium (a, overview) labeled with smooth muscle myosin S-1 and showing, in b and c, the actin filament polarity at the two ends: (b) head end and (c) trailing end. The positions of the regions enlarged in b and c are indicated in a (white arrowheads). The loss of bacterium from the head end is a typical consequence of S-1 labeling. Bars: (a) 1 μm; (b and c) 0.2 μm.
Mentions: To gain information about filament polarity, we also labeled isolated and Triton-extracted pseudopodia with myosin S-1, before fixation and negative staining. Since the labeling density was so high, it was difficult to ascertain arrowhead directions in the bulk of the tail. However, at the end of the tail, almost all filaments were oriented with their pointed ends towards the tip (Fig. 7). Interestingly, S-1 decoration commonly dislodged the bacterium from the head of the tail and revealed filaments, with few exceptions, showing barbed ends, in agreement with the data of Tilney et al. (1992a). Thus, we could confirm that the fast growing ends of the actin filaments abut the bacterium.

Bottom Line: The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin.We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane.A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg. ase@gbf-brauschweig.de

ABSTRACT
Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

Show MeSH
Related in: MedlinePlus