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Immunogold localisation of microcystins in cryosectioned cells of Microcystis
Fiona M Young1, Calcum Thomson, James S Metcalf
1Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DDI 4HN, UK. f.m.young@dundee.ac.uk
Abstract:
Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.
Insights
Cyanobacterial toxins, microcystins, were localized within Microcystis cells using immunogold labeling and cryosectioning. These toxins are primarily found associated with thylakoids and polyphosphate bodies, offering insights into their cellular roles.
Area of Science:
- Environmental Microbiology
- Cell Biology
- Toxicology
Background:
- Cyanobacterial toxins, such as microcystins, pose risks to aquatic ecosystems and human health.
- Understanding the intracellular localization of these toxins is crucial for elucidating their synthesis, function, and degradation pathways.
- Traditional sample preparation methods for electron microscopy can lead to the extraction of microcystins.
Purpose of the Study:
- To determine the precise intracellular location of microcystins within Microcystis PCC 7806 and PCC 7820 strains.
- To investigate the utility of cryosectioning for preserving microcystin integrity during electron microscopy.
- To correlate microcystin localization with cellular structures and inclusions.
Main Methods:
- Immunogold labeling was employed to visualize microcystins in laboratory cultures of Microcystis.
- Cryosectioning was utilized for immunoelectron microscopy to minimize toxin extraction during sample preparation.
- A microcystin-negative mutant strain (Microcystis PCC 7806 MC(-)) served as a control.
Main Results:
- Microcystins were specifically localized in the nucleoplasm and associated with major cellular inclusions in toxin-producing strains.
- Preferential association of microcystins was observed with thylakoids (69-78%) and polyphosphate bodies.
- Cryosectioning effectively reduced microcystin extraction compared to standard dehydration methods.
Conclusions:
- Cryosectioning is a valuable technique for accurate microcystin localization in Microcystis via electron microscopy.
- Microcystins are predominantly located in association with thylakoids and polyphosphate bodies within cyanobacterial cells.
- These findings provide critical insights into the cellular origins, functions, and fates of cyanobacterial toxins.
