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Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation.

Kokes M, Valdivia RH - PLoS ONE (2015)

Bottom Line: However, we see little evidence of intraluminal localization of these organelles in live inclusions.These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy.Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed 'inclusion') may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

No MeSH data available.


Related in: MedlinePlus

Mitochondria, ER, and inclusion membranes are found within the lumen of C. trachomatis inclusions.HeLa cells were infected with C. trachomatis LGV L2, transfected with the indicated plasmids, and fixed at 30 hpi for serial spinning disk laser confocal analysis. Note the presence of markers of the ER, mitochondrial matrix and outer membranes, and inclusion membranes within the inclusion lumen (A, cyan arrowheads). Images portray a single z-section from the center of an inclusion, and inclusions are visually identified as large black centered ovals or outlined with a dashed line. Cellular-localized markers appear saturated because material within the inclusion was often significantly dimmer. (B) The frequency of internalized structures within the entire 3D space of each inclusion was assessed. Plasmids are categorized as markers of the ER, mitochondria, inclusion, cytosol, recycling endosomes, or other as indicated. Within the other category, GFP-GalT localizes to the Golgi, CD63-GFP to MVBs, LAMP1-GFP to lysosomes, and KRphi-mRFP to the plasma membrane. A dashed line at 50% distinguishes between high and low frequencies of intraluminal structures within inclusions. 12–20 inclusions were assessed in each experiment, and the mean ± SEM for three independent experiments is shown. Scale bar represents 5 μm.
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pone.0139153.g001: Mitochondria, ER, and inclusion membranes are found within the lumen of C. trachomatis inclusions.HeLa cells were infected with C. trachomatis LGV L2, transfected with the indicated plasmids, and fixed at 30 hpi for serial spinning disk laser confocal analysis. Note the presence of markers of the ER, mitochondrial matrix and outer membranes, and inclusion membranes within the inclusion lumen (A, cyan arrowheads). Images portray a single z-section from the center of an inclusion, and inclusions are visually identified as large black centered ovals or outlined with a dashed line. Cellular-localized markers appear saturated because material within the inclusion was often significantly dimmer. (B) The frequency of internalized structures within the entire 3D space of each inclusion was assessed. Plasmids are categorized as markers of the ER, mitochondria, inclusion, cytosol, recycling endosomes, or other as indicated. Within the other category, GFP-GalT localizes to the Golgi, CD63-GFP to MVBs, LAMP1-GFP to lysosomes, and KRphi-mRFP to the plasma membrane. A dashed line at 50% distinguishes between high and low frequencies of intraluminal structures within inclusions. 12–20 inclusions were assessed in each experiment, and the mean ± SEM for three independent experiments is shown. Scale bar represents 5 μm.

Mentions: We surveyed interactions between host organelles and mid-to-late cycle inclusions by expressing a panel of fluorescent protein-tagged markers of various subcellular organelles in cells infected for 30 hr (Fig 1A). We fixed cells and quantified the frequency of cells with fluorescent material in the inclusion lumen (Fig 1B). To enhance our ability to accurately define the inclusion edge in three dimensions without interference from light above and below each plane of focus, we used confocal rather than widefield microscopy. Furthermore, since most markers had much lower intensity within the inclusion compared to cellular structures, we used spinning disk rather than laser scanning confocal microscopy to reduce photobleaching while imaging cells in three dimensions.


Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation.

Kokes M, Valdivia RH - PLoS ONE (2015)

Mitochondria, ER, and inclusion membranes are found within the lumen of C. trachomatis inclusions.HeLa cells were infected with C. trachomatis LGV L2, transfected with the indicated plasmids, and fixed at 30 hpi for serial spinning disk laser confocal analysis. Note the presence of markers of the ER, mitochondrial matrix and outer membranes, and inclusion membranes within the inclusion lumen (A, cyan arrowheads). Images portray a single z-section from the center of an inclusion, and inclusions are visually identified as large black centered ovals or outlined with a dashed line. Cellular-localized markers appear saturated because material within the inclusion was often significantly dimmer. (B) The frequency of internalized structures within the entire 3D space of each inclusion was assessed. Plasmids are categorized as markers of the ER, mitochondria, inclusion, cytosol, recycling endosomes, or other as indicated. Within the other category, GFP-GalT localizes to the Golgi, CD63-GFP to MVBs, LAMP1-GFP to lysosomes, and KRphi-mRFP to the plasma membrane. A dashed line at 50% distinguishes between high and low frequencies of intraluminal structures within inclusions. 12–20 inclusions were assessed in each experiment, and the mean ± SEM for three independent experiments is shown. Scale bar represents 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591358&req=5

pone.0139153.g001: Mitochondria, ER, and inclusion membranes are found within the lumen of C. trachomatis inclusions.HeLa cells were infected with C. trachomatis LGV L2, transfected with the indicated plasmids, and fixed at 30 hpi for serial spinning disk laser confocal analysis. Note the presence of markers of the ER, mitochondrial matrix and outer membranes, and inclusion membranes within the inclusion lumen (A, cyan arrowheads). Images portray a single z-section from the center of an inclusion, and inclusions are visually identified as large black centered ovals or outlined with a dashed line. Cellular-localized markers appear saturated because material within the inclusion was often significantly dimmer. (B) The frequency of internalized structures within the entire 3D space of each inclusion was assessed. Plasmids are categorized as markers of the ER, mitochondria, inclusion, cytosol, recycling endosomes, or other as indicated. Within the other category, GFP-GalT localizes to the Golgi, CD63-GFP to MVBs, LAMP1-GFP to lysosomes, and KRphi-mRFP to the plasma membrane. A dashed line at 50% distinguishes between high and low frequencies of intraluminal structures within inclusions. 12–20 inclusions were assessed in each experiment, and the mean ± SEM for three independent experiments is shown. Scale bar represents 5 μm.
Mentions: We surveyed interactions between host organelles and mid-to-late cycle inclusions by expressing a panel of fluorescent protein-tagged markers of various subcellular organelles in cells infected for 30 hr (Fig 1A). We fixed cells and quantified the frequency of cells with fluorescent material in the inclusion lumen (Fig 1B). To enhance our ability to accurately define the inclusion edge in three dimensions without interference from light above and below each plane of focus, we used confocal rather than widefield microscopy. Furthermore, since most markers had much lower intensity within the inclusion compared to cellular structures, we used spinning disk rather than laser scanning confocal microscopy to reduce photobleaching while imaging cells in three dimensions.

Bottom Line: However, we see little evidence of intraluminal localization of these organelles in live inclusions.These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy.Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed 'inclusion') may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

No MeSH data available.


Related in: MedlinePlus