Limits...
Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells.

Drecktrah D, Levine-Wilkinson S, Dam T, Winfree S, Knodler LA, Schroer TA, Steele-Mortimer O - Traffic (2008)

Bottom Line: Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized.Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway.Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intracellular Parasites, NIAID, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840, USA.

ABSTRACT
Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct 'leader' domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

Show MeSH

Related in: MedlinePlus

Sifs persist in the absence of microtubulesHeLa cells were infected with cherry-Salmonella and incubated for 8 h to allow for Sif formation. 37°C untreated: cells were then fixed in paraformaldehyde (PFA) for 10 min at 37°C. 37°C + nocodazole: cells were treated with nocodazole (10 μg/mL) for 15 min and then fixed in PFA with nocodazole for 10 min at 37°C. 4°C untreated: cells were shifted to ice for 15 min and fixed in PFA on ice. 4°C + nocodazole: cells were shifted to ice and treated with nocodazole for 15 min and fixed in PFA with nocodazole on ice. Subsequently, cells were processed for immunofluorescence with antibodies to LAMP1 and β-tubulin. A) Shown are z-projections of representative cells. Sif tubules were stained with anti-LAMP1 antibody (large panels), and the greyscale images have been inverted to allow for better resolution of tubules and vesicular structures. The small panels show the location of cherry-Salmonella(left and merge) and microtubules (merge). Arrowheads indicate LAMP1-positive tubules or linear arrays of LAMP1-positive vesicles. Arrows indicate the location of intracellular Salmonella. B) Quantification of Sif extension in treated cells. SEI was determined from 20 cells per treatment. Error bars represent the SD of three independent experiments. No significant difference was detected (one-way ANOVA with Dunnett's post hoctest). The mean ± SD percentage of infected cells containing Sifs for each treatment is shown in parentheses.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2682622&req=5

fig06: Sifs persist in the absence of microtubulesHeLa cells were infected with cherry-Salmonella and incubated for 8 h to allow for Sif formation. 37°C untreated: cells were then fixed in paraformaldehyde (PFA) for 10 min at 37°C. 37°C + nocodazole: cells were treated with nocodazole (10 μg/mL) for 15 min and then fixed in PFA with nocodazole for 10 min at 37°C. 4°C untreated: cells were shifted to ice for 15 min and fixed in PFA on ice. 4°C + nocodazole: cells were shifted to ice and treated with nocodazole for 15 min and fixed in PFA with nocodazole on ice. Subsequently, cells were processed for immunofluorescence with antibodies to LAMP1 and β-tubulin. A) Shown are z-projections of representative cells. Sif tubules were stained with anti-LAMP1 antibody (large panels), and the greyscale images have been inverted to allow for better resolution of tubules and vesicular structures. The small panels show the location of cherry-Salmonella(left and merge) and microtubules (merge). Arrowheads indicate LAMP1-positive tubules or linear arrays of LAMP1-positive vesicles. Arrows indicate the location of intracellular Salmonella. B) Quantification of Sif extension in treated cells. SEI was determined from 20 cells per treatment. Error bars represent the SD of three independent experiments. No significant difference was detected (one-way ANOVA with Dunnett's post hoctest). The mean ± SD percentage of infected cells containing Sifs for each treatment is shown in parentheses.

Mentions: Pretreatment of cells with the microtubule-depolymerizing agent, nocodazole, prevents Sif formation (4,14), presumably because tubule extension occurs along microtubules. However, the question of how microtubules contribute to the motility of preformed Sif tubules has never been addressed. To investigate this, we allowed Sif networks to form in infected HeLa cells for 8 h p.i. and then incubated the cells for 15 min in nocodazole (10 μg/mL) to disrupt microtubules. Live cell imaging confirmed that this treatment caused a complete cessation of movement of endocytic vesicles containing internalized fluorescent dextran, confirming that microtubules were depolymerized, and also had a dramatic effect on the dynamics of Sif tubules (Movie S2 and data not shown). Elongation and retraction ceased completely, and the Sif tubules appeared essentially ‘frozen’ in place even though microtubules were largely disassembled (Figure 6). This effect is reversible because tubule dynamics were rapidly restored following washout of nocodazole (data not shown). To confirm this result, we also used exposure to low temperature to depolymerize microtubules. In cells incubated on ice (15 min) and/or treated with nocodazole, we observed no change in either the SEI or the percentage of infected cells that contained Sif networks (Figure 6B). This result suggests that the Sif network could be stabilized by factors other than microtubules. An obvious candidate for this role is actin because, although it is not required for Sif formation (19), actin remodeling is required for intracellular replication of Salmonella in epithelial cells (20). To test this possibility, we treated cells concurrently with nocodazole and Latrunculin A (1μM), an agent that favors actin disassembly (21). Sifs persisted after this treatment as well (data not shown), indicating that their stability depends on neither actin nor microtubules.


Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells.

Drecktrah D, Levine-Wilkinson S, Dam T, Winfree S, Knodler LA, Schroer TA, Steele-Mortimer O - Traffic (2008)

Sifs persist in the absence of microtubulesHeLa cells were infected with cherry-Salmonella and incubated for 8 h to allow for Sif formation. 37°C untreated: cells were then fixed in paraformaldehyde (PFA) for 10 min at 37°C. 37°C + nocodazole: cells were treated with nocodazole (10 μg/mL) for 15 min and then fixed in PFA with nocodazole for 10 min at 37°C. 4°C untreated: cells were shifted to ice for 15 min and fixed in PFA on ice. 4°C + nocodazole: cells were shifted to ice and treated with nocodazole for 15 min and fixed in PFA with nocodazole on ice. Subsequently, cells were processed for immunofluorescence with antibodies to LAMP1 and β-tubulin. A) Shown are z-projections of representative cells. Sif tubules were stained with anti-LAMP1 antibody (large panels), and the greyscale images have been inverted to allow for better resolution of tubules and vesicular structures. The small panels show the location of cherry-Salmonella(left and merge) and microtubules (merge). Arrowheads indicate LAMP1-positive tubules or linear arrays of LAMP1-positive vesicles. Arrows indicate the location of intracellular Salmonella. B) Quantification of Sif extension in treated cells. SEI was determined from 20 cells per treatment. Error bars represent the SD of three independent experiments. No significant difference was detected (one-way ANOVA with Dunnett's post hoctest). The mean ± SD percentage of infected cells containing Sifs for each treatment is shown in parentheses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682622&req=5

fig06: Sifs persist in the absence of microtubulesHeLa cells were infected with cherry-Salmonella and incubated for 8 h to allow for Sif formation. 37°C untreated: cells were then fixed in paraformaldehyde (PFA) for 10 min at 37°C. 37°C + nocodazole: cells were treated with nocodazole (10 μg/mL) for 15 min and then fixed in PFA with nocodazole for 10 min at 37°C. 4°C untreated: cells were shifted to ice for 15 min and fixed in PFA on ice. 4°C + nocodazole: cells were shifted to ice and treated with nocodazole for 15 min and fixed in PFA with nocodazole on ice. Subsequently, cells were processed for immunofluorescence with antibodies to LAMP1 and β-tubulin. A) Shown are z-projections of representative cells. Sif tubules were stained with anti-LAMP1 antibody (large panels), and the greyscale images have been inverted to allow for better resolution of tubules and vesicular structures. The small panels show the location of cherry-Salmonella(left and merge) and microtubules (merge). Arrowheads indicate LAMP1-positive tubules or linear arrays of LAMP1-positive vesicles. Arrows indicate the location of intracellular Salmonella. B) Quantification of Sif extension in treated cells. SEI was determined from 20 cells per treatment. Error bars represent the SD of three independent experiments. No significant difference was detected (one-way ANOVA with Dunnett's post hoctest). The mean ± SD percentage of infected cells containing Sifs for each treatment is shown in parentheses.
Mentions: Pretreatment of cells with the microtubule-depolymerizing agent, nocodazole, prevents Sif formation (4,14), presumably because tubule extension occurs along microtubules. However, the question of how microtubules contribute to the motility of preformed Sif tubules has never been addressed. To investigate this, we allowed Sif networks to form in infected HeLa cells for 8 h p.i. and then incubated the cells for 15 min in nocodazole (10 μg/mL) to disrupt microtubules. Live cell imaging confirmed that this treatment caused a complete cessation of movement of endocytic vesicles containing internalized fluorescent dextran, confirming that microtubules were depolymerized, and also had a dramatic effect on the dynamics of Sif tubules (Movie S2 and data not shown). Elongation and retraction ceased completely, and the Sif tubules appeared essentially ‘frozen’ in place even though microtubules were largely disassembled (Figure 6). This effect is reversible because tubule dynamics were rapidly restored following washout of nocodazole (data not shown). To confirm this result, we also used exposure to low temperature to depolymerize microtubules. In cells incubated on ice (15 min) and/or treated with nocodazole, we observed no change in either the SEI or the percentage of infected cells that contained Sif networks (Figure 6B). This result suggests that the Sif network could be stabilized by factors other than microtubules. An obvious candidate for this role is actin because, although it is not required for Sif formation (19), actin remodeling is required for intracellular replication of Salmonella in epithelial cells (20). To test this possibility, we treated cells concurrently with nocodazole and Latrunculin A (1μM), an agent that favors actin disassembly (21). Sifs persisted after this treatment as well (data not shown), indicating that their stability depends on neither actin nor microtubules.

Bottom Line: Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized.Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway.Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intracellular Parasites, NIAID, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840, USA.

ABSTRACT
Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct 'leader' domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

Show MeSH
Related in: MedlinePlus