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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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Video sequences showing Listeria-induced pseudopodium behavior in PtK2 cells (a) and macrophages (b). Time is indicated  in min. (a) One bacterium is already in a pseudopodium (1) at time 0′, and the second (2) is in bulk cytoplasm at the head of a comet tail.  The pseudopodium (1) gyrates actively for several minutes, and then reenters the main body of the cell at around 5′, forming a typical  comet tail (6′ and 7′). The second bacterium begins to form a pseudopodium at 2′, which then exists from 3′ to 5′, after which the bacterium reenters the cell (at 6′), immediately forming a comet (7′). (b) Two pseudopodia (1 and 2) are shown that have already extended  away from the macrophage cell body (bottom righthand corner) and continue to move during the sequence, being tethered to the cell by  a thin strand. Conditions: (a) phase contrast; (b) Normarski interference contrast. Bars: (a and b) 4.5 μm.
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Figure 1: Video sequences showing Listeria-induced pseudopodium behavior in PtK2 cells (a) and macrophages (b). Time is indicated in min. (a) One bacterium is already in a pseudopodium (1) at time 0′, and the second (2) is in bulk cytoplasm at the head of a comet tail. The pseudopodium (1) gyrates actively for several minutes, and then reenters the main body of the cell at around 5′, forming a typical comet tail (6′ and 7′). The second bacterium begins to form a pseudopodium at 2′, which then exists from 3′ to 5′, after which the bacterium reenters the cell (at 6′), immediately forming a comet (7′). (b) Two pseudopodia (1 and 2) are shown that have already extended away from the macrophage cell body (bottom righthand corner) and continue to move during the sequence, being tethered to the cell by a thin strand. Conditions: (a) phase contrast; (b) Normarski interference contrast. Bars: (a and b) 4.5 μm.

Mentions: Fig. 1 a shows a video series of an infected PtK2 cell in which the fate of two comet tails can be followed. The first (Fig. 1 a, 1) already existed as a pseudopodium for 3 min before the beginning of the sequence and actively waved around in the medium for an additional 4 min before reentering the parent cell and immediately continuing its movement. The second (Fig. 1 a, 2), started in the main body of the cell, formed a pseudopodium, and then reinvaded the cell within the sequence, spending only 2 min as a pseudopodium. For this latter type of protrusion, the velocity of movement of the bacterium in the protruding phase was the same as in the bulk cytoplasm. Measurements showed the average lifetime of pseudopodia to be ∼3 min ± 1 min (15 measurements), and the rate of movement inside growing pseudopodia averaged 0.074 μm/s (SD ± 0.01 μm/s) as compared with a rate of 0.15 μm/s (SD ± 0.04 μm/s) for bacteria freely moving in the cytoplasm.


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)

Video sequences showing Listeria-induced pseudopodium behavior in PtK2 cells (a) and macrophages (b). Time is indicated  in min. (a) One bacterium is already in a pseudopodium (1) at time 0′, and the second (2) is in bulk cytoplasm at the head of a comet tail.  The pseudopodium (1) gyrates actively for several minutes, and then reenters the main body of the cell at around 5′, forming a typical  comet tail (6′ and 7′). The second bacterium begins to form a pseudopodium at 2′, which then exists from 3′ to 5′, after which the bacterium reenters the cell (at 6′), immediately forming a comet (7′). (b) Two pseudopodia (1 and 2) are shown that have already extended  away from the macrophage cell body (bottom righthand corner) and continue to move during the sequence, being tethered to the cell by  a thin strand. Conditions: (a) phase contrast; (b) Normarski interference contrast. Bars: (a and b) 4.5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139863&req=5

Figure 1: Video sequences showing Listeria-induced pseudopodium behavior in PtK2 cells (a) and macrophages (b). Time is indicated in min. (a) One bacterium is already in a pseudopodium (1) at time 0′, and the second (2) is in bulk cytoplasm at the head of a comet tail. The pseudopodium (1) gyrates actively for several minutes, and then reenters the main body of the cell at around 5′, forming a typical comet tail (6′ and 7′). The second bacterium begins to form a pseudopodium at 2′, which then exists from 3′ to 5′, after which the bacterium reenters the cell (at 6′), immediately forming a comet (7′). (b) Two pseudopodia (1 and 2) are shown that have already extended away from the macrophage cell body (bottom righthand corner) and continue to move during the sequence, being tethered to the cell by a thin strand. Conditions: (a) phase contrast; (b) Normarski interference contrast. Bars: (a and b) 4.5 μm.
Mentions: Fig. 1 a shows a video series of an infected PtK2 cell in which the fate of two comet tails can be followed. The first (Fig. 1 a, 1) already existed as a pseudopodium for 3 min before the beginning of the sequence and actively waved around in the medium for an additional 4 min before reentering the parent cell and immediately continuing its movement. The second (Fig. 1 a, 2), started in the main body of the cell, formed a pseudopodium, and then reinvaded the cell within the sequence, spending only 2 min as a pseudopodium. For this latter type of protrusion, the velocity of movement of the bacterium in the protruding phase was the same as in the bulk cytoplasm. Measurements showed the average lifetime of pseudopodia to be ∼3 min ± 1 min (15 measurements), and the rate of movement inside growing pseudopodia averaged 0.074 μm/s (SD ± 0.01 μm/s) as compared with a rate of 0.15 μm/s (SD ± 0.04 μm/s) for bacteria freely moving in the cytoplasm.

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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Related in: MedlinePlus