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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.

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Imaging of Sifs loaded with endocytosed dextransA) HeLa cells were loaded with fluorescent dextrans and infected with cherry-Salmonella as described in the text (for clarity, a graphic depicting the experimental scheme is shown). Cells were then imaged using a spinning disk confocal microscope. A single processed image from a time-lapse series is shown. N, nucleus. Arrowheads indicate tubules containing both fluorescent dextrans. Arrows indicate the location of intracellular Salmonella. B) To visualize delivery of incoming endocytic content to Sifs, cells were infected with Salmonella and dex-AF488 was added as a 30-min pulse at 6 h p.i. as depicted in the graphic showing the experimental scheme. A series of images is shown; elapsed time is indicated. Between 2.8 and 5.6 seconds, a dex-AF488-loaded vesicle (large arrowhead) can be seen delivering its content to a pre-existing Sif tubule (indicated with small arrowheads) (see also Movie S3). Scale bar = 10 μm. C) Quantification of the transient (contact; black bar) versus long-term (>20 seconds; docking; white bar) interactions of endosomes with Sif tubules as a function of distance from the end of the tubule. Values are mean ± SEM. Because leader Sifs are much dimmer than vesicles, they could not be readily seen in this analysis, so the distance measurements are assumed to be relative to the end of the trailing Sif domain. Asterisk indicates that the mean distances are significantly different.
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fig07: Imaging of Sifs loaded with endocytosed dextransA) HeLa cells were loaded with fluorescent dextrans and infected with cherry-Salmonella as described in the text (for clarity, a graphic depicting the experimental scheme is shown). Cells were then imaged using a spinning disk confocal microscope. A single processed image from a time-lapse series is shown. N, nucleus. Arrowheads indicate tubules containing both fluorescent dextrans. Arrows indicate the location of intracellular Salmonella. B) To visualize delivery of incoming endocytic content to Sifs, cells were infected with Salmonella and dex-AF488 was added as a 30-min pulse at 6 h p.i. as depicted in the graphic showing the experimental scheme. A series of images is shown; elapsed time is indicated. Between 2.8 and 5.6 seconds, a dex-AF488-loaded vesicle (large arrowhead) can be seen delivering its content to a pre-existing Sif tubule (indicated with small arrowheads) (see also Movie S3). Scale bar = 10 μm. C) Quantification of the transient (contact; black bar) versus long-term (>20 seconds; docking; white bar) interactions of endosomes with Sif tubules as a function of distance from the end of the tubule. Values are mean ± SEM. Because leader Sifs are much dimmer than vesicles, they could not be readily seen in this analysis, so the distance measurements are assumed to be relative to the end of the trailing Sif domain. Asterisk indicates that the mean distances are significantly different.

Mentions: Sifs emanate from a parent compartment, the SCV, which is accessible to incoming endocytic markers and can fuse directly with lysosomes containing fluorescently labeled dextrans as soluble content markers (2). In this study, we used a similar approach to investigate whether Sif tubules are similarly able to fuse with components of the endocytic pathway. Cells were incubated overnight with Alexa Fluor 647-conjugated dextran (dex-AF647), then infected with cherry-Salmonella and incubated for an additional 6 h in the presence of dex-AF647. Dex-AF647 was then removed, and cells were incubated for two more hours in the presence of a second color dextran, dex-AF488. Live cell imaging was initiated as soon as the excess dextran was removed by washing. Under these conditions, almost all the Sif tubules we observed contained both fluorescent dextrans (Figure 7), indicating that Sif biogenesis involves significant interactions with endocytic organelles. Because dex-AF647 was present continuously during the entire initial period of Sif formation, it may have been delivered to Sifs or the SCV from any element of the endocytic pathway. However, that dex-AF488 that had been taken up by cells starting at 6 h p.i. was also able to gain access to Sifs suggests that early and/or late endosomes might be able to deliver content to tubules by fusing with them directly. To test this hypothesis, we internalized dex-AF488 for a brief (30 min) pulse after Sifs were allowed to form and then looked for fusion events by live cell imaging. As shown in Figure 7B and Figure S3, dex-AF488-loaded endosomes were observed fusing with pre-existing Sif tubules and delivering their contents into the tubule lumen. These experiments demonstrate unequivocally that Sif tubules are accessible to, and in direct communication with, incoming endocytic traffic.


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)

Imaging of Sifs loaded with endocytosed dextransA) HeLa cells were loaded with fluorescent dextrans and infected with cherry-Salmonella as described in the text (for clarity, a graphic depicting the experimental scheme is shown). Cells were then imaged using a spinning disk confocal microscope. A single processed image from a time-lapse series is shown. N, nucleus. Arrowheads indicate tubules containing both fluorescent dextrans. Arrows indicate the location of intracellular Salmonella. B) To visualize delivery of incoming endocytic content to Sifs, cells were infected with Salmonella and dex-AF488 was added as a 30-min pulse at 6 h p.i. as depicted in the graphic showing the experimental scheme. A series of images is shown; elapsed time is indicated. Between 2.8 and 5.6 seconds, a dex-AF488-loaded vesicle (large arrowhead) can be seen delivering its content to a pre-existing Sif tubule (indicated with small arrowheads) (see also Movie S3). Scale bar = 10 μm. C) Quantification of the transient (contact; black bar) versus long-term (>20 seconds; docking; white bar) interactions of endosomes with Sif tubules as a function of distance from the end of the tubule. Values are mean ± SEM. Because leader Sifs are much dimmer than vesicles, they could not be readily seen in this analysis, so the distance measurements are assumed to be relative to the end of the trailing Sif domain. Asterisk indicates that the mean distances are significantly different.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682622&req=5

fig07: Imaging of Sifs loaded with endocytosed dextransA) HeLa cells were loaded with fluorescent dextrans and infected with cherry-Salmonella as described in the text (for clarity, a graphic depicting the experimental scheme is shown). Cells were then imaged using a spinning disk confocal microscope. A single processed image from a time-lapse series is shown. N, nucleus. Arrowheads indicate tubules containing both fluorescent dextrans. Arrows indicate the location of intracellular Salmonella. B) To visualize delivery of incoming endocytic content to Sifs, cells were infected with Salmonella and dex-AF488 was added as a 30-min pulse at 6 h p.i. as depicted in the graphic showing the experimental scheme. A series of images is shown; elapsed time is indicated. Between 2.8 and 5.6 seconds, a dex-AF488-loaded vesicle (large arrowhead) can be seen delivering its content to a pre-existing Sif tubule (indicated with small arrowheads) (see also Movie S3). Scale bar = 10 μm. C) Quantification of the transient (contact; black bar) versus long-term (>20 seconds; docking; white bar) interactions of endosomes with Sif tubules as a function of distance from the end of the tubule. Values are mean ± SEM. Because leader Sifs are much dimmer than vesicles, they could not be readily seen in this analysis, so the distance measurements are assumed to be relative to the end of the trailing Sif domain. Asterisk indicates that the mean distances are significantly different.
Mentions: Sifs emanate from a parent compartment, the SCV, which is accessible to incoming endocytic markers and can fuse directly with lysosomes containing fluorescently labeled dextrans as soluble content markers (2). In this study, we used a similar approach to investigate whether Sif tubules are similarly able to fuse with components of the endocytic pathway. Cells were incubated overnight with Alexa Fluor 647-conjugated dextran (dex-AF647), then infected with cherry-Salmonella and incubated for an additional 6 h in the presence of dex-AF647. Dex-AF647 was then removed, and cells were incubated for two more hours in the presence of a second color dextran, dex-AF488. Live cell imaging was initiated as soon as the excess dextran was removed by washing. Under these conditions, almost all the Sif tubules we observed contained both fluorescent dextrans (Figure 7), indicating that Sif biogenesis involves significant interactions with endocytic organelles. Because dex-AF647 was present continuously during the entire initial period of Sif formation, it may have been delivered to Sifs or the SCV from any element of the endocytic pathway. However, that dex-AF488 that had been taken up by cells starting at 6 h p.i. was also able to gain access to Sifs suggests that early and/or late endosomes might be able to deliver content to tubules by fusing with them directly. To test this hypothesis, we internalized dex-AF488 for a brief (30 min) pulse after Sifs were allowed to form and then looked for fusion events by live cell imaging. As shown in Figure 7B and Figure S3, dex-AF488-loaded endosomes were observed fusing with pre-existing Sif tubules and delivering their contents into the tubule lumen. These experiments demonstrate unequivocally that Sif tubules are accessible to, and in direct communication with, incoming endocytic traffic.

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