Correlative light and electron microscopy harnesses the best from each of the two modalities of m... more Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope.
The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have... more The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) corelate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group...
Expansion microscopy is an imaging method based on isotropic physical expansion of biological sam... more 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 non-adherent cells and multiciliated cultures.
Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, an... more Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, and its mutations are found in patients with diseases such as primary microcephaly. CPAP’s centrosomal localization, its dynamics, and the consequences of its insufficiency in human cells are poorly understood. Here we use human cells genetically engineered for fast degradation of CPAP, in combination with superresolution microscopy, to address these uncertainties. We show that three independent centrosomal CPAP populations are dynamically regulated during the cell cycle. We confirm that CPAP is critical for assembly of human centrioles, but not for recruitment of pericentriolar material on already assembled centrioles. Further, we reveal that CPAP insufficiency leads to centrioles with incomplete microtubule triplets that can convert to centrosomes, duplicate, and form mitotic spindle poles, but fragment owing to loss of cohesion between microtubule blades. These findings further our basi...
SummaryCentrioles are vital cellular structures that organise centrosomes and cilia. Due to their... more SummaryCentrioles 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 DescriptionCentrioles are microtubule‐based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 n...
Correlative light and electron microscopy harnesses the best from each of the two modalities of m... more Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope.
The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have... more The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) corelate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group...
Expansion microscopy is an imaging method based on isotropic physical expansion of biological sam... more 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 non-adherent cells and multiciliated cultures.
Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, an... more Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, and its mutations are found in patients with diseases such as primary microcephaly. CPAP’s centrosomal localization, its dynamics, and the consequences of its insufficiency in human cells are poorly understood. Here we use human cells genetically engineered for fast degradation of CPAP, in combination with superresolution microscopy, to address these uncertainties. We show that three independent centrosomal CPAP populations are dynamically regulated during the cell cycle. We confirm that CPAP is critical for assembly of human centrioles, but not for recruitment of pericentriolar material on already assembled centrioles. Further, we reveal that CPAP insufficiency leads to centrioles with incomplete microtubule triplets that can convert to centrosomes, duplicate, and form mitotic spindle poles, but fragment owing to loss of cohesion between microtubule blades. These findings further our basi...
SummaryCentrioles are vital cellular structures that organise centrosomes and cilia. Due to their... more SummaryCentrioles 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 DescriptionCentrioles are microtubule‐based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 n...
Correlative light and electron microscopy harnesses the best from each of the two modalities of m... more Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope.
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