Building Cell Structures in Three Dimensions: Electron Tomography Methods for Budding Yeast.

has been an important model system for numerous cellular, genetic, and molecular studies. However, this small eukaryote presents a challenge for imaging at the electron microscope level. Preparation of yeast using high-pressure freezing followed by freeze-substitution (HPF/FS) results in excellent preservation of cell structure in these difficult-to-fix samples.

Cryopreparation and Electron Tomography of Yeast Cells.

Three-dimensional imaging of cells using electron tomography enables analysis of cell structure at unprecedented resolution. The preparation of cells for tomography using rapid freezing followed by freeze-substitution is an essential first step to ensure the optimal preservation of the cell structure for 3D studies. This protocol outlines a method for obtaining well-preserved cells using high-pressure freezing followed by freeze-substitution.

Electron Microscopy of Fission Yeast.

Electron microscopy (EM) can provide images of cells with a spatial resolution that significantly surpasses that available from light microscopy (LM), even with modern methods that give LM "super resolution." However, EM resolution comes with costs in time spent with sample preparation, expense of instrumentation, and concerns regarding sample preparation artifacts. It is therefore important to know the limitations of EM as well as its strengths.

Cryoelectron Microscopy of Fission Yeast.

Fission yeast cells can be prepared for electron microscopy (EM) in the frozen-hydrated state. This eliminates the requirement for dehydration and heavy metal staining when preparing samples for EM. As with room temperature imaging, however, the yeast must be sectioned to make them thin enough for transmission of the electron beam.

Immunolocalization of Proteins in Fission Yeast by Electron Microscopy.

Electron microscopy (EM) immunolocalization of antigens in fission yeast can be accomplished with cells processed by rapid freezing and freeze-substitution followed by embedding in acrylic or methacrylate resins. Microtome sections of embedded cells are collected onto EM grids. Primary antibodies to the antigen of interest, followed by secondary antibodies conjugated to colloidal gold, are allowed to bind to antigens at the surface of these plastic sections.

Preparing Fission Yeast for Electron Microscopy.

Freezing samples while simultaneously subjecting them to a rapid increase in pressure, which inhibits ice crystal formation, is a reliable method for cryofixing fission yeast. The procedure consists simply of harvesting cells and loading them into a high-pressure freezer (HPF), and then operating the device. If equipment for high-pressure freezing is not available, fission yeast can be frozen by plunging a monolayer of cells into a liquid cryogen, usually ethane or propane.

Tetrahymena Poc1 ensures proper intertriplet microtubule linkages to maintain basal body integrity.

Basal bodies comprise nine symmetric triplet microtubules that anchor forces produced by the asymmetric beat pattern of motile cilia. The ciliopathy protein Poc1 stabilizes basal bodies through an unknown mechanism. In poc1∆ cells, electron tomography reveals subtle defects in the organization of intertriplet linkers (A-C linkers) that connect adjacent triplet microtubules.

The negatively charged carboxy-terminal tail of β-tubulin promotes proper chromosome segregation.

Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and β-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating β-CTT causes elevated rates of chromosome loss and cell cycle delay.

A Short CEP135 Splice Isoform Controls Centriole Duplication.

Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1-5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that, when centriole protein expression and/or activity are increased, centrioles self-assemble.

Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning.

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain.

DisAp-dependent striated fiber elongation is required to organize ciliary arrays.

Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1.

Membrane dynamics at the nuclear exchange junction during early mating (one to four hours) in the ciliate Tetrahymena thermophila.

Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane.

Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast.

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication.

Conventional transmission electron microscopy.

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design.

ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies.

Basal bodies and centrioles are conserved microtubule-based organelles the improper assembly of which leads to a number of diseases, including ciliopathies and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. We have identified the ε-tubulin gene in Tetrahymena thermophila and detected the protein, through fluorescence of a tagged allele, to basal bodies.

Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol.

Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces.

Basal bodies nucleate, anchor, and organize cilia. As the anchor for motile cilia, basal bodies must be resistant to the forces directed toward the cell as a consequence of ciliary beating. The molecules and generalized mechanisms that contribute to the maintenance of basal bodies remain to be discovered.

The two human centrin homologues have similar but distinct functions at Tetrahymena basal bodies.

Centrins are a ubiquitous family of small Ca(2+)-binding proteins found at basal bodies that are placed into two groups based on sequence similarity to the human centrins 2 and 3. Analyses of basal body composition in different species suggest that they contain a centrin isoform from each group. We used the ciliate protist Tetrahymena thermophila to gain a better understanding of the functions of the two centrin groups and to determine their potential redundancy.

Computer-assisted image analysis of human cilia and Chlamydomonas flagella reveals both similarities and differences in axoneme structure.

In the past decade, investigations from several different fields have revealed the critical role of cilia in human health and disease. Because of the highly conserved nature of the basic axonemal structure, many different model systems have proven useful for the study of ciliopathies, especially the unicellular, biflagellate green alga Chlamydomonas reinhardtii. Although the basic axonemal structure of cilia and flagella is highly conserved, these organelles often perform specialized functions unique to the cell or tissue in which they are found.

Cytological analysis of Tetrahymena thermophila.

Since their first detection in pond water, large ciliates such as Tetrahymena thermophila, have captivated school children and scientists alike with the elegance of their swimming and the beauty of their cortical organization. Indeed, cytology - simply looking at cells - is an important component of most areas of study in cell biology and is particularly intriguing in the large, complex Tetrahymena cell. Cytological analysis of Tetrahymena is critical for the study of the microtubule cytoskeleton, membrane trafficking, complex nuclear movements and interactions, and the cellular remodeling during conjugation, to name a few topics.

Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii.

Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome.

The Aurora kinase Ipl1 is necessary for spindle pole body cohesion during budding yeast meiosis.

In budding yeast, the microtubule-organizing center is called the spindle pole body (SPB) and shares structural components with the centriole, the central core of the animal centrosome. During meiotic interphase I, the SPB is duplicated when DNA replication takes place. Duplicated SPBs are linked and then separate to form a bipolar spindle required for homolog separation in meiosis I.

The two domains of centrin have distinct basal body functions in Tetrahymena.

The basal body is a microtubule-organizing center responsible for organizing the cilium, a structure important for cell locomotion and sensing of the surrounding environment. A widely conserved basal body component is the Ca(2+)-binding protein centrin. Analyses of centrin function suggest a role in basal body assembly and stability; however, its molecular mechanisms remain unclear.

Electron tomography and immuno-labeling of Tetrahymena thermophila basal bodies.

Basal bodies and centrioles are highly ordered, microtubule-based organelles involved in the organization of the mitotic spindle and the formation of cilia and flagella. The ciliate Tetrahymena thermophila has more than 700 basal bodies per cell, making it an excellent choice for the study of the structure, function, and assembly of basal bodies. Here, we describe methods for cryofixation of Tetrahymena by high-pressure freezing and freeze-substitution (HPF/FS) for the analysis of basal body structure with advanced electron microscopy techniques.