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Analyzing Centrioles and Cilia by Expansion Microscopy.

ABSTRACT: Expansion microscopy is an imaging method based on isotropic physical expansion of biological samples, which improves optical resolution and allows imaging of subresolutional cellular components by conventional microscopes. Centrioles are small microtubule-based cylindrical structures that build centrosomes and cilia, two organelles essential for vertebrates. Due to a centriole's small size, electron microscopy has traditionally been used to study centriole length and ultrastructural features. Recently, expansion microscopy has been successfully used as an affordable and accessible alternative to electron microscopy in the analysis of centriole and cilia length and structural features. Here, we describe an expansion microscopy approach for the analysis of centrioles and cilia in large populations of mammalian adherent and nonadherent cells and multiciliated cultures.

SUBMITTER: Kong D

PROVIDER: S-EPMC8344367 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Analyzing Centrioles and Cilia by Expansion Microscopy.

Kong Dong D Loncarek Jadranka J

Methods in molecular biology (Clifton, N.J.) 20210101

Expansion microscopy is an imaging method based on isotropic physical expansion of biological samples, which improves optical resolution and allows imaging of subresolutional cellular components by conventional microscopes. Centrioles are small microtubule-based cylindrical structures that build centrosomes and cilia, two organelles essential for vertebrates. Due to a centriole's small size, electron microscopy has traditionally been used to study centriole length and ultrastructural features. R ...[more]

PMID: 34085228

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Project description:Centrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures. LAY DESCRIPTION: Centrioles are microtubule-based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 nm wide. Centrioles assemble dozens of proteins around them forming centrosomes, which nucleate microtubules and organise spindle poles in mitosis. Centrioles, in addition, assemble cilia and flagella, two critically important organelles for signalling and motility. Due to centriole small size, electron microscopy has been a major imaging technique for the analysis of their ultrastructural features. However, being technically demanding, electron microscopy it is not easily available to the researchers and it is rarely used to collect large datasets. Expansion microscopy is an emerging approach in which biological specimens are embedded in a swellable polymer and isotopically expanded several fold. Physical separation of cellular structures allows the analysis of, otherwise unresolvable, structures by conventional optical microscopes. We present an adaptation of expansion microscopy approach, specifically developed for a robust analysis of centrioles and cilia. Our protocol can be used for the analysis of centriole number, duplication status, length, localisation of various centrosomal components and ciliation from large populations of cultured adherent and nonadherent cells and multiciliated cultures. We validate the method against electron microscopy and superresolution microscopy and demonstrate that it can be used as an accessible and reliable alternative to electron microscopy.

Dataset Information

Analyzing Centrioles and Cilia by Expansion Microscopy.

Publications

Analyzing Centrioles and Cilia by Expansion Microscopy.

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