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Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities.

ABSTRACT: Transmission electron microscopy (TEM) can provide ultrastructural information for cells in microbial community samples and phylogenetic information can be recovered via molecular surveys. Here we report an approach to link these data sets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM. The method could fundamentally improve our understanding of the organization and functioning of microbial communities in natural systems.

SUBMITTER: Knierim B

PROVIDER: S-EPMC4444221 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities.

Knierim Bernhard B Luef Birgit B Wilmes Paul P Webb Richard I RI Auer Manfred M Comolli Luis R LR Banfield Jillian F JF

Environmental microbiology reports 20110822 1

Transmission electron microscopy (TEM) can provide ultrastructural information for cells in microbial community samples and phylogenetic information can be recovered via molecular surveys. Here we report an approach to link these data sets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM. The method could fundamentally improve our understanding of the organization and functioning of microbial communities in natural systems. ...[more]

PMID: 23757227

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Project description:Podosomes are multimolecular cytoskeletal structures that coordinate the migration of tissue-resident dendritic cells (DCs). They consist of a protrusive actin-rich core and an adhesive integrin-rich ring that contains adaptor proteins such as vinculin and zyxin. Individual podosomes are typically interconnected by a dense network of actin filaments giving rise to large podosome clusters. The actin density in podosome clusters complicates the analysis of podosomes by light microscopy alone. Here, we present an optimized procedure for performing super-resolution correlative light and electron microscopy (SR-CLEM) to study the organization of multiple proteins with respect to actin in podosome clusters at the ventral plasma membrane of DCs. We demonstrate that our procedure is suited to correlate at least three colors in super-resolution Airyscan microscopy with scanning electron microscopy (SEM). Using this procedure, we first reveal an intriguing complexity in the organization of ventral and radiating actin filaments in clusters formed by DCs which was not properly detected before by light microscopy alone. Next, we demonstrate a differential organization of vinculin and zyxin with respect to the actin filaments at podosomes. While vinculin mostly resides at sites where the actin filaments connect to the cell membrane, zyxin is primarily associated with filaments close to and on top of the core. Finally, we reveal a novel actin-based structure with SEM that connects closely associated podosome cores and which may be important for podosome topography sensing. Interestingly, these interpodosomal connections, in contrast to the radiating and ventral actin filaments appear to be insensitive to inhibition of actin polymerization suggesting that these pools of actin are not dynamically coupled. Together, our work demonstrates the power of correlating different imaging modalities for studying multimolecular cellular structures and could potentially be further exploited to study processes at the ventral plasma membrane of immune cells such as clathrin-mediated endocytosis or immune synapse formation.

Project description:BackgroundThe infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria.ResultsThe liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria.ConclusionsA CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Dataset Information

Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities.

Publications

Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities.

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