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Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Denoncourt AM, Paquet VE, Sedighi A, Charette SJ - PLoS ONE (2016)

Bottom Line: The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody.Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody.This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, Canada.

ABSTRACT
Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

No MeSH data available.


Related in: MedlinePlus

Comparison of the anti-SctA and H36 antibodies as MLB markers.Immunofluorescence analyses of purified MLBs using the (A) anti-SctA and (B) H36 antibodies. SctA labeling was not uniform and the protein appeared to be concentrated in aggregates in the MLBs. H36 labeling was only seen on the surface of MLBs, creating a ring-like appearance. The purified MLBs were deposited on glass slides and were processed for immunofluorescence using the anti-SctA or H36 antibodies. (C) Flow cytometric analysis of purified MLBs labeled with the anti-SctA or H36 antibody. Approximately 40% of the MLBs were labeled with the anti-SctA antibody while 60–70% were labeled with the H36 antibody. The fluorescence of the labeled MLBs was much more intense with the H36 antibody than with the anti-SctA antibody. Scale bar: A and B = 5 μm.
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pone.0158270.g005: Comparison of the anti-SctA and H36 antibodies as MLB markers.Immunofluorescence analyses of purified MLBs using the (A) anti-SctA and (B) H36 antibodies. SctA labeling was not uniform and the protein appeared to be concentrated in aggregates in the MLBs. H36 labeling was only seen on the surface of MLBs, creating a ring-like appearance. The purified MLBs were deposited on glass slides and were processed for immunofluorescence using the anti-SctA or H36 antibodies. (C) Flow cytometric analysis of purified MLBs labeled with the anti-SctA or H36 antibody. Approximately 40% of the MLBs were labeled with the anti-SctA antibody while 60–70% were labeled with the H36 antibody. The fluorescence of the labeled MLBs was much more intense with the H36 antibody than with the anti-SctA antibody. Scale bar: A and B = 5 μm.

Mentions: The H36 antibody may be a more suitable MLB marker than the anti-SctA antibody. The efficiency of the two antibodies as MLB markers was compared by immunofluorescence and flow cytometry. Fig 5 shows fluorescent microscopic images of purified MLBs labeled with the anti-SctA (A) and H36 (B) antibodies. In the case of the anti-SctA antibody, the labeling was not uniform and appeared as dots scattered throughout the MLBs. In the case of the H36 antibody, the labeling remained at the surface of the MLBs, giving them a ring-like appearance. The fluorescent signal was less intense for the anti-SctA antibody than for the H36 antibody.


Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Denoncourt AM, Paquet VE, Sedighi A, Charette SJ - PLoS ONE (2016)

Comparison of the anti-SctA and H36 antibodies as MLB markers.Immunofluorescence analyses of purified MLBs using the (A) anti-SctA and (B) H36 antibodies. SctA labeling was not uniform and the protein appeared to be concentrated in aggregates in the MLBs. H36 labeling was only seen on the surface of MLBs, creating a ring-like appearance. The purified MLBs were deposited on glass slides and were processed for immunofluorescence using the anti-SctA or H36 antibodies. (C) Flow cytometric analysis of purified MLBs labeled with the anti-SctA or H36 antibody. Approximately 40% of the MLBs were labeled with the anti-SctA antibody while 60–70% were labeled with the H36 antibody. The fluorescence of the labeled MLBs was much more intense with the H36 antibody than with the anti-SctA antibody. Scale bar: A and B = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920372&req=5

pone.0158270.g005: Comparison of the anti-SctA and H36 antibodies as MLB markers.Immunofluorescence analyses of purified MLBs using the (A) anti-SctA and (B) H36 antibodies. SctA labeling was not uniform and the protein appeared to be concentrated in aggregates in the MLBs. H36 labeling was only seen on the surface of MLBs, creating a ring-like appearance. The purified MLBs were deposited on glass slides and were processed for immunofluorescence using the anti-SctA or H36 antibodies. (C) Flow cytometric analysis of purified MLBs labeled with the anti-SctA or H36 antibody. Approximately 40% of the MLBs were labeled with the anti-SctA antibody while 60–70% were labeled with the H36 antibody. The fluorescence of the labeled MLBs was much more intense with the H36 antibody than with the anti-SctA antibody. Scale bar: A and B = 5 μm.
Mentions: The H36 antibody may be a more suitable MLB marker than the anti-SctA antibody. The efficiency of the two antibodies as MLB markers was compared by immunofluorescence and flow cytometry. Fig 5 shows fluorescent microscopic images of purified MLBs labeled with the anti-SctA (A) and H36 (B) antibodies. In the case of the anti-SctA antibody, the labeling was not uniform and appeared as dots scattered throughout the MLBs. In the case of the H36 antibody, the labeling remained at the surface of the MLBs, giving them a ring-like appearance. The fluorescent signal was less intense for the anti-SctA antibody than for the H36 antibody.

Bottom Line: The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody.Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody.This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, Canada.

ABSTRACT
Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

No MeSH data available.


Related in: MedlinePlus