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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

ER markers reveal expansive structures within the inclusion lumen of fixed but not living cells.HeLa cells were infected with C. trachomatis LGV L2, co-transfected with ER-RFP (red) and Sec61β-GFP (green) and at 30 hpi were either fixed or imaged in three dimensions while living. Images were acquired with a laser scanning confocal microscope. (A) A single xy micrograph towards the center of a cell. See S1 and S2 Movies for a progression of xy micrographs along the z-axis. (B) Images were used to render volumes in 3D. 3D render of a fixed cell is limited in the z-axis to allow viewing within the inclusion. See S3–S6 Movies for QuickTime Virtual Reality files to rotate and view these 3D volumes. Note the presence of an expansive network of material within the inclusion lumen of fixed cells. N marks the nucleus. Scale bars represent 5 μm.
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pone.0139153.g002: ER markers reveal expansive structures within the inclusion lumen of fixed but not living cells.HeLa cells were infected with C. trachomatis LGV L2, co-transfected with ER-RFP (red) and Sec61β-GFP (green) and at 30 hpi were either fixed or imaged in three dimensions while living. Images were acquired with a laser scanning confocal microscope. (A) A single xy micrograph towards the center of a cell. See S1 and S2 Movies for a progression of xy micrographs along the z-axis. (B) Images were used to render volumes in 3D. 3D render of a fixed cell is limited in the z-axis to allow viewing within the inclusion. See S3–S6 Movies for QuickTime Virtual Reality files to rotate and view these 3D volumes. Note the presence of an expansive network of material within the inclusion lumen of fixed cells. N marks the nucleus. Scale bars represent 5 μm.

Mentions: The protein markers that were most notably visible within the lumen of inclusions, such as ER-RFP and Sec61β-GFP, typically formed an expansive network of large blebs and tubules within much of the three-dimensional space of the inclusion lumen in fixed cells (Fig 2A and 2B, top panel, S1, S3, and S4 Movies). However, within the limits of our experimental design and number of cells analyzed, we were unable to detect these structures within living infected cells (Fig 2A and 2B, bottom panel, S2, S5 and S6 Movies).


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

Kokes M, Valdivia RH - PLoS ONE (2015)

ER markers reveal expansive structures within the inclusion lumen of fixed but not living cells.HeLa cells were infected with C. trachomatis LGV L2, co-transfected with ER-RFP (red) and Sec61β-GFP (green) and at 30 hpi were either fixed or imaged in three dimensions while living. Images were acquired with a laser scanning confocal microscope. (A) A single xy micrograph towards the center of a cell. See S1 and S2 Movies for a progression of xy micrographs along the z-axis. (B) Images were used to render volumes in 3D. 3D render of a fixed cell is limited in the z-axis to allow viewing within the inclusion. See S3–S6 Movies for QuickTime Virtual Reality files to rotate and view these 3D volumes. Note the presence of an expansive network of material within the inclusion lumen of fixed cells. N marks the nucleus. Scale bars represent 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591358&req=5

pone.0139153.g002: ER markers reveal expansive structures within the inclusion lumen of fixed but not living cells.HeLa cells were infected with C. trachomatis LGV L2, co-transfected with ER-RFP (red) and Sec61β-GFP (green) and at 30 hpi were either fixed or imaged in three dimensions while living. Images were acquired with a laser scanning confocal microscope. (A) A single xy micrograph towards the center of a cell. See S1 and S2 Movies for a progression of xy micrographs along the z-axis. (B) Images were used to render volumes in 3D. 3D render of a fixed cell is limited in the z-axis to allow viewing within the inclusion. See S3–S6 Movies for QuickTime Virtual Reality files to rotate and view these 3D volumes. Note the presence of an expansive network of material within the inclusion lumen of fixed cells. N marks the nucleus. Scale bars represent 5 μm.
Mentions: The protein markers that were most notably visible within the lumen of inclusions, such as ER-RFP and Sec61β-GFP, typically formed an expansive network of large blebs and tubules within much of the three-dimensional space of the inclusion lumen in fixed cells (Fig 2A and 2B, top panel, S1, S3, and S4 Movies). However, within the limits of our experimental design and number of cells analyzed, we were unable to detect these structures within living infected cells (Fig 2A and 2B, bottom panel, S2, S5 and S6 Movies).

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