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. We have found that this method is particularly well suited for electron tomography studies and has the added bonus of preserving antigenicity for immuno-electron microscopy. The steps involved in imaging cells and performing tomographic analysis of cellular structures are also outlined.

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

Saccharomyces cerevisiae 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. In particular, cells prepared by HPF/FS can be used for 3D electron tomography (ET) studies where optimum cell preservation is critical. Here, we discuss the advantages of using HPF/FS for ET and show examples of the utility of this method for building yeast cell structures in three dimensions.

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. Unlike the HPF, where relatively large volumes of cells can be frozen in a single run, plunge freezing requires cells to be dispersed in a layer <20 µm thick. Unless frozen cells are to be imaged in the vitreous state, they must be fixed, dehydrated, and embedded for subsequent study by transmission electron microscopy; warming frozen cells without fixation badly damages cell structure. Fixation is best accomplished by freeze-substitution, a process in which frozen water is removed from samples by a water-miscible solvent that is liquid at a temperature low enough to prevent the cellular water from recrystallizing. Low concentrations of chemical fixatives and stains are generally added to this solvent such that they permeate the cells as the water is replaced. The activity of these additives is quite limited at the low temperatures required for minimizing ice crystal formation, but they are in the right place to react effectively as the cells warm up. Step-by-step protocols for HPF, plunge freezing, and freeze-substitution are provided here.

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. This type of postembed labeling provides information on antigen localization to a resolution of 10-20 nm, depending on the size of the metal particle used, the form of the antibody (Fab vs. complete IgG or IgM), and whether direct or indirect labeling is used. The method has the potential to map macromolecules in three dimensions in a relatively large volume when thin (30-60-nm) serial sections are labeled, imaged, aligned, and modeled to create a representative volume. The biggest challenge of this technique is the necessary compromise between the preservation of cellular ultrastructure and the preservation of antigen reactivity. The protocols described here show how to immunolabel samples for EM and include suggestions for overcoming challenges related to antigen preservation.

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. Cutting sections of vitreous ice with a microtome is challenging. An alternative method that uses a focused ion beam to make a thin sample by milling away much of the sample at liquid nitrogen temperatures is under development but is not yet available for routine use. Imaging frozen-hydrated samples by EM is also a challenge. The technique involves battling low image contrast, high sensitivity to the electron beam, and mechanical distortions produced during the sectioning process. When used successfully, however, the method holds promise of providing excellent molecular detail without the disruption characteristic of dehydration or isolating a structure from its cellular environment. Cryo-EM of tilted views can be used to examine small structures and macromolecular complexes in their native cellular environment. If a structure exists in multiple copies, or has a repeating unit, it can be investigated at higher resolution using subvolume averaging. This protocol focuses on the preparation of cells for cryo-EM.

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. Here we describe the most reliable methods for the preservation of fission yeast cells currently available. We describe the properties of images obtained by transmission EM (TEM) and contrast them with images from scanning EM (SEM). We also show how one can make three-dimensional TEM images and discuss several approaches to address the problem of localizing specific proteins within cells. We give references to work by others who have pursued similar goals with different methods, and we discuss briefly the complex subject of image interpretation.

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. Complete triplet microtubules are lost preferentially near the posterior face of the basal body. Basal bodies that are missing triplets likely remain competent to assemble new basal bodies with nine triplet microtubules, suggesting that the mother basal body microtubule structure does not template the daughter. Our data indicate that Poc1 stabilizes basal body triplet microtubules through linkers between neighboring triplets. Without this stabilization, specific triplet microtubules within the basal body are more susceptible to loss, probably due to force distribution within the basal body during ciliary beating. This work provides insights into how the ciliopathy protein Poc1 maintains basal body integrity.

The negatively charged carboxy-terminal tail of ß-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 ß-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating ß-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that ß-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that ß-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles.

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. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6-11]. SAS-6 constitutes the cartwheel, and SAS-6 levels remain low until centriole assembly is initiated at S phase onset [3, 12, 13]. CEP135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of CEP135 that antagonize each other to modulate centriole duplication: full-length CEP135 (CEP135(full)) promotes new assembly, whereas a short isoform, CEP135(mini), represses it. CEP135(mini) represses centriole duplication by limiting the centriolar localization of CEP135(full) binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The CEP135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. CEP135(mini) protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in CEP135(mini) from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

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. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.

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. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.

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. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp's similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

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. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.

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. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.

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. Immunoelectron microscopy has shown that ε-tubulin localizes primarily to the core microtubule scaffold. A complete genomic knockout of ε-tubulin has revealed that it is an essential gene required for the assembly and maintenance of the triplet microtubule blades of basal bodies. We have conducted site-directed mutagenesis of the ε-tubulin gene and shown that residues within the nucleotide-binding domain, longitudinal interacting domains, and C-terminal tail are required for proper function. A single amino acid change of Thr150, a conserved residue in the nucleotide-binding domain, to Val is a conditional mutation that results in defects in the spatial and temporal assembly of basal bodies as well as their stability. We have genetically separated functions for the domains of ε-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.

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. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.

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. Bld10/Cep135 is a basal body outer cartwheel domain protein that has established roles in the assembly of nascent basal bodies. We find that Bld10 protein first incorporates stably at basal bodies early during new assembly. Bld10 protein continues to accumulate at basal bodies after assembly, and we hypothesize that the full complement of Bld10 is required to stabilize basal bodies. We identify a novel mechanism for Bld10/Cep135 in basal body maintenance so that basal bodies can withstand the forces produced by motile cilia. Bld10 stabilizes basal bodies by promoting the stability of the A- and C-tubules of the basal body triplet microtubules and by properly positioning the triplet microtubule blades. The forces generated by ciliary beating promote basal body disassembly in bld10Δ cells. Thus Bld10/Cep135 acts to maintain the structural integrity of basal bodies against the forces of ciliary beating in addition to its separable role in basal body assembly.

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. We have previously shown that the Tetrahymena centrin 1 (Cen1), a human centrin 2 homologue, is required for proper basal body function. In this paper, we show that the Tetrahymena centrin 2 (Cen2), a human centrin 3 homologue, has functions similar to Cen1 in basal body orientation, maintenance, and separation. The two are, however, not redundant. A further examination of human centrin 3 homologues shows that they function in a manner distinct from human centrin 2 homologues. Our data suggest that basal bodies require a centrin from both groups in order to function correctly.

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. These differences in function are likely reflected in differences in structural organization. In this work, we directly compare the structure of isolated axonemes from human cilia and Chlamydomonas flagella to identify similarities and differences that potentially play key roles in determining their functionality. Using transmission electron microscopy and 2D image averaging techniques, our analysis has confirmed the overall structural similarity between these two species, but also revealed clear differences in the structure of the outer dynein arms, the central pair projections, and the radial spokes. We also show how the application of 2D image averaging can clarify the underlying structural defects associated with primary ciliary dyskinesia (PCD). Overall, our results document the remarkable similarity between these two structures separated evolutionarily by over a billion years, while highlighting several significant differences, and demonstrate the potential of 2D image averaging to improve the diagnosis and understanding of PCD.