The bacterial cytoskeleton: more than twisted filaments

Far from being simple ‘bags’ of enzymes, bacteria are richly endowed with ultrastructures that challenge and expand standard definitions of the cytoskeleton. Here we review rods, rings, twisted pairs, tubes, sheets, spirals, moving patches, meshes and composites, and suggest defining the term ‘bacterial cytoskeleton’ as all cytoplasmic protein filaments and their superstructures that move or scaffold (stabilize/position/recruit) other cellular materials. The evolution of each superstructure has been driven by specific functional requirements. As a result, while homologous proteins with different functions have evolved to form surprisingly divergent superstructures, those of unrelated proteins with similar functions have converged

A new view into prokaryotic cell biology from electron cryotomography

Electron cryotomography (ECT) enables intact cells to be visualized in 3D in an essentially native state to 'macromolecular' (~4 nm) resolution, revealing the basic architectures of complete nanomachines and their arrangements in situ. Since its inception, ECT has advanced our understanding of many aspects of prokaryotic cell biology, from morphogenesis to subcellular compartmentalization and from metabolism to complex interspecies interactions. In this Review, we highlight how ECT has provided structural and mechanistic insights into the physiology of bacteria and archaea and discuss prospects for the future

Rapid Tilt-Series Method for Cryo-Electron Tomography: Characterizing Stage Behavior During FISE Acquisition

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals

Organization of the Smallest Eukaryotic Spindle

In metazoans, plants, and fungi, the spindle checkpoint delays mitosis until each chromosome is attached to one or more of its own kinetochore microtubules (kMTs). Some unicellular eukaryotes, however, have been reported to have fewer kMTs than chromosomes. If this is the case, it is unclear how the spindle checkpoint could be satisfied. In the vast majority of the previous studies, mitotic cells were chemically fixed at room temperature, but this does not always preserve dynamic and/or small structures like spindle MTs and kinetochores. Indeed, later higher-resolution studies have reversed some earlier claims. Here we show that in Ostreococcus tauri (the smallest eukaryote known), mitosis does involve fewer spindle microtubules than chromosomes. O. tauri cultures were enriched for mitotic cells, high-pressure frozen, and then imaged in 3D both in plastic and in a near-native ("frozen-hydrated") state through electron tomography. Mitotic cells have a distinctive intranuclear heterochromatin-free "spindle tunnel" with approximately four short and occasionally one long, incomplete (unclosed) microtubule at each end of the spindle tunnel. Because other aspects of O. tauri’s spindle checkpoint seem typical, these data suggest that O. tauri’s 20 chromosomes are physically linked and segregated as just one or a small number of groups

Electron Cryotomography

Electron cryotomography (ECT) is an emerging technology that allows thin samples such as macromolecular complexes and small bacterial cells to be imaged in 3-D in a nearly native state to “molecular” (~4 nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal filaments, cell wall elements, motility machines, chemoreceptor arrays, internal compartments, and other ultrastructures. This article describes the technique and summarizes its contributions to bacterial cell biology. For comparable recent reviews, see (Subramaniam 2005; Jensen and Briegel 2007; Murphy and Jensen 2007; Li and Jensen 2009). For reviews on the history, technical details, and broader application of electron tomography in general, see for example (Subramaniam and Milne 2004; Lucić et al. 2005; Leis et al. 2008; Midgley and Dunin-Borkowski 2009)