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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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(a) General organization of actin filaments in pseudopodia. Long filaments provide the axis of the tail, and short filaments cross-link them (circles) into a loose bundle in the region  proximal to the bacterium. The comet tails are presumed to have  the same structure as the pseudopodia, except that they feature  more randomly arranged short filaments. In the more distal parts  of the tail, where there are fewer short filaments, the cross-linking between axial filaments predominates (not indicated). (b)  Schematic illustration of how the short and long filaments may be  generated. The rear end of the bacterium is depicted at different  stages of forward movement (arrow) at times t1–t4. The grey, external layer houses components of the polymerization machinery  that nucleates actin filaments and feeds their barbed (+) ends  with actin monomers. When a filament (+) end lies outside this  polymerization zone, it is capped by host cell capping factors  (squares). For clarity, only a few actin filaments (straight lines)  are shown. Arrowhead configurations indicate the pointed (−)  and barbed (+) ends of the filaments. The lengths of a filament  will be determined by the position on the rear of the bacterium at  which it becomes nucleated and the orientation to the membrane  adopted at that time (we assume this to be variable), since this  will define how long the filament can reside in the polymerization  zone as the bacterium moves forward. The ends of filaments 1  and 4 fall out of the influence of the polymerization zone at t2, after they become tangential to its outer surface. They will be short  and obliquely oriented to the tail axis. For filament 2, polymerization ceases at t3. By the same token, more axially oriented filaments (3 and 5) will maintain their plus ends in the polymerization zone for a more extended period and thus become  correspondingly longer. L. cell wall, Listeria cell wall.
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Figure 8: (a) General organization of actin filaments in pseudopodia. Long filaments provide the axis of the tail, and short filaments cross-link them (circles) into a loose bundle in the region proximal to the bacterium. The comet tails are presumed to have the same structure as the pseudopodia, except that they feature more randomly arranged short filaments. In the more distal parts of the tail, where there are fewer short filaments, the cross-linking between axial filaments predominates (not indicated). (b) Schematic illustration of how the short and long filaments may be generated. The rear end of the bacterium is depicted at different stages of forward movement (arrow) at times t1–t4. The grey, external layer houses components of the polymerization machinery that nucleates actin filaments and feeds their barbed (+) ends with actin monomers. When a filament (+) end lies outside this polymerization zone, it is capped by host cell capping factors (squares). For clarity, only a few actin filaments (straight lines) are shown. Arrowhead configurations indicate the pointed (−) and barbed (+) ends of the filaments. The lengths of a filament will be determined by the position on the rear of the bacterium at which it becomes nucleated and the orientation to the membrane adopted at that time (we assume this to be variable), since this will define how long the filament can reside in the polymerization zone as the bacterium moves forward. The ends of filaments 1 and 4 fall out of the influence of the polymerization zone at t2, after they become tangential to its outer surface. They will be short and obliquely oriented to the tail axis. For filament 2, polymerization ceases at t3. By the same token, more axially oriented filaments (3 and 5) will maintain their plus ends in the polymerization zone for a more extended period and thus become correspondingly longer. L. cell wall, Listeria cell wall.

Mentions: Inasmuch as the pseudopodia are motile and can revert rapidly into their parent comet tails on reentry into bulk cytoplasm (e.g., Fig. 1 a), we can presume that the basic mechanism of movement of Listeria in the main body of the cell and in the pseudopodia is identical. Changes do, however, occur as the comet tails enter the protrusion phase; α-actinin is lost and there is a decrease in total F-actin, as judged by the intensity of phalloidin staining. We propose that the decrease in actin cross-linking that presumably results from the loss of α-actinin leads to the shedding of a sizeable proportion of the short filaments from the comet tail as it forms a pseudopodium. Dold et al. (1994) have indeed shown that the comet tails are disrupted in cells microinjected with a gelation-incompetent fragment of α-actinin, suggesting that this protein here plays a major role in filament cross-linking. A reduction of cross-linking would at the same time facilitate a collimation of the longitudinal filaments into a more compact bundle, as seen in the isolated pseudopodia. In this context, the short filaments are seen as structural elements required for long distance, lateral cross-linking within the comet tail (Fig. 8 a) and between the tail and the surrounding cytoskeleton. In the realm of bulk cytoplasm, such a function is vital to restrain the borders of the tail and to give it polarity and support, but it becomes of lesser importance in the membranecoated pseudopodia. Dabiri et al. (1990) have previously claimed that α-actinin is present in pseudopodia, but inspection of their images reveals that the intensity of fluorescent label in pseudopodia was the same as in the nonspecific background in the cell.


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)

(a) General organization of actin filaments in pseudopodia. Long filaments provide the axis of the tail, and short filaments cross-link them (circles) into a loose bundle in the region  proximal to the bacterium. The comet tails are presumed to have  the same structure as the pseudopodia, except that they feature  more randomly arranged short filaments. In the more distal parts  of the tail, where there are fewer short filaments, the cross-linking between axial filaments predominates (not indicated). (b)  Schematic illustration of how the short and long filaments may be  generated. The rear end of the bacterium is depicted at different  stages of forward movement (arrow) at times t1–t4. The grey, external layer houses components of the polymerization machinery  that nucleates actin filaments and feeds their barbed (+) ends  with actin monomers. When a filament (+) end lies outside this  polymerization zone, it is capped by host cell capping factors  (squares). For clarity, only a few actin filaments (straight lines)  are shown. Arrowhead configurations indicate the pointed (−)  and barbed (+) ends of the filaments. The lengths of a filament  will be determined by the position on the rear of the bacterium at  which it becomes nucleated and the orientation to the membrane  adopted at that time (we assume this to be variable), since this  will define how long the filament can reside in the polymerization  zone as the bacterium moves forward. The ends of filaments 1  and 4 fall out of the influence of the polymerization zone at t2, after they become tangential to its outer surface. They will be short  and obliquely oriented to the tail axis. For filament 2, polymerization ceases at t3. By the same token, more axially oriented filaments (3 and 5) will maintain their plus ends in the polymerization zone for a more extended period and thus become  correspondingly longer. L. cell wall, Listeria cell wall.
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Related In: Results  -  Collection

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Figure 8: (a) General organization of actin filaments in pseudopodia. Long filaments provide the axis of the tail, and short filaments cross-link them (circles) into a loose bundle in the region proximal to the bacterium. The comet tails are presumed to have the same structure as the pseudopodia, except that they feature more randomly arranged short filaments. In the more distal parts of the tail, where there are fewer short filaments, the cross-linking between axial filaments predominates (not indicated). (b) Schematic illustration of how the short and long filaments may be generated. The rear end of the bacterium is depicted at different stages of forward movement (arrow) at times t1–t4. The grey, external layer houses components of the polymerization machinery that nucleates actin filaments and feeds their barbed (+) ends with actin monomers. When a filament (+) end lies outside this polymerization zone, it is capped by host cell capping factors (squares). For clarity, only a few actin filaments (straight lines) are shown. Arrowhead configurations indicate the pointed (−) and barbed (+) ends of the filaments. The lengths of a filament will be determined by the position on the rear of the bacterium at which it becomes nucleated and the orientation to the membrane adopted at that time (we assume this to be variable), since this will define how long the filament can reside in the polymerization zone as the bacterium moves forward. The ends of filaments 1 and 4 fall out of the influence of the polymerization zone at t2, after they become tangential to its outer surface. They will be short and obliquely oriented to the tail axis. For filament 2, polymerization ceases at t3. By the same token, more axially oriented filaments (3 and 5) will maintain their plus ends in the polymerization zone for a more extended period and thus become correspondingly longer. L. cell wall, Listeria cell wall.
Mentions: Inasmuch as the pseudopodia are motile and can revert rapidly into their parent comet tails on reentry into bulk cytoplasm (e.g., Fig. 1 a), we can presume that the basic mechanism of movement of Listeria in the main body of the cell and in the pseudopodia is identical. Changes do, however, occur as the comet tails enter the protrusion phase; α-actinin is lost and there is a decrease in total F-actin, as judged by the intensity of phalloidin staining. We propose that the decrease in actin cross-linking that presumably results from the loss of α-actinin leads to the shedding of a sizeable proportion of the short filaments from the comet tail as it forms a pseudopodium. Dold et al. (1994) have indeed shown that the comet tails are disrupted in cells microinjected with a gelation-incompetent fragment of α-actinin, suggesting that this protein here plays a major role in filament cross-linking. A reduction of cross-linking would at the same time facilitate a collimation of the longitudinal filaments into a more compact bundle, as seen in the isolated pseudopodia. In this context, the short filaments are seen as structural elements required for long distance, lateral cross-linking within the comet tail (Fig. 8 a) and between the tail and the surrounding cytoskeleton. In the realm of bulk cytoplasm, such a function is vital to restrain the borders of the tail and to give it polarity and support, but it becomes of lesser importance in the membranecoated pseudopodia. Dabiri et al. (1990) have previously claimed that α-actinin is present in pseudopodia, but inspection of their images reveals that the intensity of fluorescent label in pseudopodia was the same as in the nonspecific background in the cell.

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