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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 PtK2 pseudopodium (a) indicating regions (bracketed) shown at higher magnification in b and c. The component of  axial filaments in b is best revealed by viewing the micrograph at a glancing angle. Bars: (a) 1 μm; (b and c) 0.2 μm.
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Figure 3: Isolated PtK2 pseudopodium (a) indicating regions (bracketed) shown at higher magnification in b and c. The component of axial filaments in b is best revealed by viewing the micrograph at a glancing angle. Bars: (a) 1 μm; (b and c) 0.2 μm.

Mentions: In line with the differences noted in the light microscope, pseudopodia isolated from PtK2 cells showed subtle differences in structure to those from the macrophage cell line. First of all, they differed in length, the isolated PtK2 pseudopodia averaging 7 ± 2.4 μm and those from macrophages 16.5 ± 4.7 μm. But most notably, PtK2 pseudopodia characteristically exhibited more randomly organized short filaments throughout their length, especially in the region proximal to the bacterium (Fig. 3). Measurements showed the random filaments to fall in the length range of 0.3–1.0 μm, with a mean of 0.57 ± 0.17 μm. In addition to these randomly oriented filaments, a set of long filaments aligned along the axis of the tail was also evident. These filaments were somewhat dispersed in the proximity of the bacterium and became progressively bundled towards the midpart and end of the tail. Fig. 4 shows a thin PtK2 pseudopod that spread out laterally on the support film, revealing rather clearly the two sets of actin filaments, random and longitudinal. In this example the continuity of the longitudinally arranged filaments is especially evident and can be emphasized by tilting the micrograph to view the filaments at a grazing angle. Single filaments could be traced for up to 2 μm along their length, before being lost in the more densely packed regions.


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 PtK2 pseudopodium (a) indicating regions (bracketed) shown at higher magnification in b and c. The component of  axial filaments in b is best revealed by viewing the micrograph at a glancing angle. 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 3: Isolated PtK2 pseudopodium (a) indicating regions (bracketed) shown at higher magnification in b and c. The component of axial filaments in b is best revealed by viewing the micrograph at a glancing angle. Bars: (a) 1 μm; (b and c) 0.2 μm.
Mentions: In line with the differences noted in the light microscope, pseudopodia isolated from PtK2 cells showed subtle differences in structure to those from the macrophage cell line. First of all, they differed in length, the isolated PtK2 pseudopodia averaging 7 ± 2.4 μm and those from macrophages 16.5 ± 4.7 μm. But most notably, PtK2 pseudopodia characteristically exhibited more randomly organized short filaments throughout their length, especially in the region proximal to the bacterium (Fig. 3). Measurements showed the random filaments to fall in the length range of 0.3–1.0 μm, with a mean of 0.57 ± 0.17 μm. In addition to these randomly oriented filaments, a set of long filaments aligned along the axis of the tail was also evident. These filaments were somewhat dispersed in the proximity of the bacterium and became progressively bundled towards the midpart and end of the tail. Fig. 4 shows a thin PtK2 pseudopod that spread out laterally on the support film, revealing rather clearly the two sets of actin filaments, random and longitudinal. In this example the continuity of the longitudinally arranged filaments is especially evident and can be emphasized by tilting the micrograph to view the filaments at a grazing angle. Single filaments could be traced for up to 2 μm along their length, before being lost in the more densely packed regions.

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