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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.


MLBs have different morphologies.Transmission electron microscopic images of MLBs composed of (A) concentric membrane layers, (B) a mix of concentric membrane layers and amorphous material, and (C) amorphous material. Scale bar: A to C = 0.5 μm.
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pone.0158270.g003: MLBs have different morphologies.Transmission electron microscopic images of MLBs composed of (A) concentric membrane layers, (B) a mix of concentric membrane layers and amorphous material, and (C) amorphous material. Scale bar: A to C = 0.5 μm.

Mentions: In the presence of K. aerogenes, D. discoideum produced SctA-positive extracellular MLBs, which can be distinguished from pycnosomes by their larger size (Fig 2B). Several MLBs were detected by differential interference contrast microscopy (DIC) but not all of them were SctA-positive. SctA was also observed in dot-like structures inside the amoebae. Purified MLBs are shown in Fig 2C. While most of the structures appeared to be SctA positive, the intensity of the fluorescence was uneven. This might explain why some of the MLBs (produced in the presence of K. aerogenes) shown in Fig 2B were not SctA-positive. In addition, in samples containing amoebae, bacteria, and debris, it was harder to distinguish the weak signal of MLBs with fewer SctA proteins. The SctA labeling was also concentrated in dots rather than being spread out uniformly in the MLBs. It is possible that more or less compact pycnosomes are embedded in the MLBs, which would explain the faint labeling of MLBs by the anti-SctA antibody. Indeed, some MLBs exhibited regions of non-concentric membrane layers (Fig 3), which are similar to the pycnosome structures inside the cells (See Sabra et al. [17]). It has been shown that SctA-positive structures, likely pycnosomes, are incorporated into intraendosomal membranes in cells treated with U18666A [17], a drug that induces the formation of multilamellar structures in endosomes [6].


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

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

MLBs have different morphologies.Transmission electron microscopic images of MLBs composed of (A) concentric membrane layers, (B) a mix of concentric membrane layers and amorphous material, and (C) amorphous material. Scale bar: A to C = 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920372&req=5

pone.0158270.g003: MLBs have different morphologies.Transmission electron microscopic images of MLBs composed of (A) concentric membrane layers, (B) a mix of concentric membrane layers and amorphous material, and (C) amorphous material. Scale bar: A to C = 0.5 μm.
Mentions: In the presence of K. aerogenes, D. discoideum produced SctA-positive extracellular MLBs, which can be distinguished from pycnosomes by their larger size (Fig 2B). Several MLBs were detected by differential interference contrast microscopy (DIC) but not all of them were SctA-positive. SctA was also observed in dot-like structures inside the amoebae. Purified MLBs are shown in Fig 2C. While most of the structures appeared to be SctA positive, the intensity of the fluorescence was uneven. This might explain why some of the MLBs (produced in the presence of K. aerogenes) shown in Fig 2B were not SctA-positive. In addition, in samples containing amoebae, bacteria, and debris, it was harder to distinguish the weak signal of MLBs with fewer SctA proteins. The SctA labeling was also concentrated in dots rather than being spread out uniformly in the MLBs. It is possible that more or less compact pycnosomes are embedded in the MLBs, which would explain the faint labeling of MLBs by the anti-SctA antibody. Indeed, some MLBs exhibited regions of non-concentric membrane layers (Fig 3), which are similar to the pycnosome structures inside the cells (See Sabra et al. [17]). It has been shown that SctA-positive structures, likely pycnosomes, are incorporated into intraendosomal membranes in cells treated with U18666A [17], a drug that induces the formation of multilamellar structures in endosomes [6].

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.