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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-RFP translocates into the inclusion lumen during chemical fixation.HeLa cells were infected with C. trachomatis LGV L2 and transfected with ER-RFP. At 30 hpi, living cells were placed in 4% paraformaldehyde and imaged over time with a laser scanning confocal microscope. Note the genesis of ER-RFP blebs into the inclusions lumen (cyan arrowheads) during fixation. See S7 Movie for a time-lapse video including more cells in a larger field of view. N marks the nuclei. Scale bar represents 10 μm.
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pone.0139153.g003: ER-RFP translocates into the inclusion lumen during chemical fixation.HeLa cells were infected with C. trachomatis LGV L2 and transfected with ER-RFP. At 30 hpi, living cells were placed in 4% paraformaldehyde and imaged over time with a laser scanning confocal microscope. Note the genesis of ER-RFP blebs into the inclusions lumen (cyan arrowheads) during fixation. See S7 Movie for a time-lapse video including more cells in a larger field of view. N marks the nuclei. Scale bar represents 10 μm.

Mentions: Because of the differences in ER localization patterns between living and fixed cells, we considered the possibility that the process of fixation exaggerates the degree of translocation of this organelle into the inclusion lumen. To test this, we used laser scanning confocal microscopy on living infected cells to monitor changes to ER-RFP localization during paraformaldehyde fixation (Fig 3 and S7 Movie). Within minutes of the addition of fixative, we observed the formation of blebs of ER-RFP material expanding into the inclusion lumen. These blebs appeared at random sites along the inclusion periphery and enlarged over time as new sites of inward blebbing (Fig 3, arrowheads) emerged. Many blebs remained attached to the inclusion edge but some appeared to detach into the center of the inclusion. By ten minutes, most new ER blebbing and expansion had stopped and existing structures varied in fluorescence intensity. We also noted the formation of a few smaller and less distinct bleb-like aggregates of ER-RFP in other areas of the cell, mostly at cell edges. These findings demonstrate that chemical fixation can induce a dramatic translocation of ER material into the inclusion lumen.


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

Kokes M, Valdivia RH - PLoS ONE (2015)

ER-RFP translocates into the inclusion lumen during chemical fixation.HeLa cells were infected with C. trachomatis LGV L2 and transfected with ER-RFP. At 30 hpi, living cells were placed in 4% paraformaldehyde and imaged over time with a laser scanning confocal microscope. Note the genesis of ER-RFP blebs into the inclusions lumen (cyan arrowheads) during fixation. See S7 Movie for a time-lapse video including more cells in a larger field of view. N marks the nuclei. Scale bar represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139153.g003: ER-RFP translocates into the inclusion lumen during chemical fixation.HeLa cells were infected with C. trachomatis LGV L2 and transfected with ER-RFP. At 30 hpi, living cells were placed in 4% paraformaldehyde and imaged over time with a laser scanning confocal microscope. Note the genesis of ER-RFP blebs into the inclusions lumen (cyan arrowheads) during fixation. See S7 Movie for a time-lapse video including more cells in a larger field of view. N marks the nuclei. Scale bar represents 10 μm.
Mentions: Because of the differences in ER localization patterns between living and fixed cells, we considered the possibility that the process of fixation exaggerates the degree of translocation of this organelle into the inclusion lumen. To test this, we used laser scanning confocal microscopy on living infected cells to monitor changes to ER-RFP localization during paraformaldehyde fixation (Fig 3 and S7 Movie). Within minutes of the addition of fixative, we observed the formation of blebs of ER-RFP material expanding into the inclusion lumen. These blebs appeared at random sites along the inclusion periphery and enlarged over time as new sites of inward blebbing (Fig 3, arrowheads) emerged. Many blebs remained attached to the inclusion edge but some appeared to detach into the center of the inclusion. By ten minutes, most new ER blebbing and expansion had stopped and existing structures varied in fluorescence intensity. We also noted the formation of a few smaller and less distinct bleb-like aggregates of ER-RFP in other areas of the cell, mostly at cell edges. These findings demonstrate that chemical fixation can induce a dramatic translocation of ER material into the inclusion lumen.

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