Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of nature’s most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy

Unclosed HIV-1 Capsids Suggest a Curled Sheet Model of Assembly

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~ 25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms

In Situ Imaging of Bacterial Outer Membrane Projections and Associated Protein Complexes Using Electron Cryo-Tomography

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field

Uncharacterized bacterial structures revealed by electron cryotomography

Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells

The Structural Biology of HIV Budding and Maturation

The Human Immunodeficiency Virus (HIV) depends on the ability to exit infected cells, mature into an infectious state, and infect new host cells. The structural details of exiting and maturation (known as the "late stage events") remain elusive, but further understanding could lead to new therapies. HIV exits cells by hijacking a host cellular complex called ESCRT (Endosomal Sorting Complex Required for Transport), which evolved to constrict membranes in multivesicular body formation and cytokinesis. Electron cryotomography (ECT) was used to gain three-dimensional images of ESCRT in several contexts, including the physiological system of archaeal cell division. This study provided insight into the monomer interactions in the complex and led to a molecular mechanism of membrane constriction. HIV is released from the cell as an immature particle with the main structural protein, Gag, forming a spherical shell around the RNA genome and enzymes. Gag is then cleaved into several proteins that refold and assemble into the conical capsid that is characteristic of the mature, infectious particle. The capsid is typically a closed cone, but unclosed varieties provide insight to the mechanism of assembly. By combining ECT, computer simulations, and fluorescence light microscopy, we analyzed features of unclosed capsids that suggest a "curling sheet" model of capsid assembly. These studies not only provided novel insight into the late stages of the HIV life cycle, but also contributed to the methods used by electron microscopists and researchers of HIV.</p

The Structure, Function and Roles of the Archaeal ESCRT Apparatus

Although morphologically resembling bacteria, archaea constitute a distinct domain of life with a closer affiliation to eukaryotes than to bacteria. This similarity is seen in the machineries for a number of essential cellular processes, including DNA replication and gene transcription. Perhaps surprisingly, given their prokaryotic morphology, some archaea also possess a core cell division apparatus that is related to that involved in the final stages of membrane abscission in vertebrate cells, the ESCRT machinery

Plunge Freezing for Electron Cryomicroscopy

Aqueous biological samples must be “preserved” (stabilized) before they can be placed in the high vacuum of an electron microscope. Among the various approaches that have been developed, plunge freezing maintains the sample in the most native state and is therefore the method of choice when possible. Plunge freezing for standard electron cryomicroscopy applications proceeds by spreading the sample into a thin film across an EM grid and then rapidly submerging it in a cryogen (usually liquid ethane), but success depends critically on the properties of the grid and sample, the production of a uniformly thin film, the temperature and nature of the cryogen, and the plunging conditions. This chapter reviews plunge-freezing principles, techniques, instrumentation, common problems, and safety considerations

<i>Ambystoma jeffersonianum-laterale</i> embryos.

<p>A) Typical <i>Ambystoma jeffersonianum-laterale</i> eggs in cleavage stage 4 laid in a pond in western Massachusetts. B) A portion of a clutch extracted from a road-killed unisexual <i>Ambystoma jeffersonianum-laterale</i>. C) Fluorescence image of a DAPI-stained embryo from the clutch of the road-killed individual in approximately development stage 10. The blue dots are cell nuclei, the pink dots are surface reflections of the light source. D) A sectioned embryo showing a fluid-filled center with nuclei distributed around the periphery. E) A close up of nuclei in an embryo.</p