Microbial Cell Biology, HIV, and the Development of Electron Cryotomography
Summary: Grant Jensen's laboratory uses state-of-the-art electron cryotomographical techniques to understand the structure and function of large protein machines and their arrangement within cells. Projects range from theoretical studies on the mathematics of three-dimensional reconstructions and testing of new instrumentation to high-resolution 3D imaging of viruses and cells in near-native states.
Microbial Cell Biology
Although thousands of bacterial genomes have now been sequenced and a variety of high-throughput "omic" technologies are revealing gene expression patterns and how the various gene products mediate metabolism, our comparative ignorance about the more physical and mechanical processes that occur in a bacterial life cycle is surprising. We still do not know, for instance, how bacteria generate and maintain their characteristic shapes, establish polarity, organize their genomes, segregate their chromosomes, divide, and in some cases move. In some sense, the "omic" technologies are giving us lists of parts and reactions, but bacterial cells are not merely bags of enzymes—structural and mechanical details are also needed.
Fortunately, the emergence of electron cryotomography has now allowed us to obtain unprecedented three-dimensional (3D) views of proteins, viruses, and even whole bacterial cells in near-native states to molecular (~2- to 5-nm) resolution. In our lab we are both helping to develop electron cryotomography as a structural technique and also using it to study the structures and functions of the supramolecular complexes that, for example, shape bacterial cells, propel them forward, sense where to swim, organize metabolism, secrete signals, attack competitors, and drive growth and division.
The prokaryotic cytoskeleton. Because for many decades cytoskeletal filaments were only rarely seen in traditional thin-section electron microscopy (EM) images of bacteria, it was long thought that prokaryotes lacked a cytoskeleton and that this was one of the hallmark differences between prokaryotes and eukaryotes. More recently, however, bacterial homologs of actin, tubulin, and intermediate filament proteins have been identified and fluorescent light microscopy (fLM) has confirmed that these and other proteins sometimes localize in filament-like patterns in vivo. We and others have now used cryotomography to directly visualize many different cytoskeletal filaments and filament bundles in a variety of species, confirming that bacteria do indeed have elaborate and extensive cytoskeletal systems. Seeing these filaments for the first time, however, is generating as many questions as answers: what is each filament, which fLM results were authentic and which were artifacts, and what is each filament doing? Cryotomography is helping us answer these questions.
Cell shape. The characteristic shapes of bacteria are established and maintained by a supramolecular "bag" or "sacculus" of peptidoglycan that completely surrounds the cell. Through direct imaging we have elucidated the basic architecture of the peptidoglycan cell wall in both Gram-positive and Gram-negative cells and shown that sporulation involves interconversion between the two.
Motility machines. Most motile bacteria propel themselves with flagella. We published the first structure of an entire flagellar motor, which showed, among other things, the number and shape of the stator "studs" in situ. We have now reconstructed motors from several different species; these comparisons highlight how each species has adapted the motor to its purposes. The Treponema primitia motor, for example, is wider in diameter and has more stator studs, adaptations that could "gear" it down to produce the higher torque it might need as a spirochete to rotate the entire cell. Nonflagellar (gliding) motility mechanisms in bacteria have also been described. We imaged the attachment organelle of Mycoplasma pneumoniae and proposed that it is a conformationally dynamic engine that drives gliding motility in that species.
Chemotaxis. Given a means to move, bacteria must also know where to go. Bacterial chemotaxis is mediated by an array of chemoreceptors embedded in the cell membrane that send signals to the flagellar motors. By fitting x-ray crystal structures into high-resolution cryotomographical maps we generated the first pseudoatomic model of a chemoreceptor array, revealing that the receptors are arranged as a hexagonal lattice of trimers of receptor dimers. We also showed that the basic architecture of chemoreceptor arrays is conserved across bacteria, and we are now exploring the structural basis of array activation.
Secretion, pathogenesis, and competition. Bacteria have evolved a number of molecular machines to secrete wastes, kill competitors, and inject regulatory molecules into host cells. Through direct imaging, we recently discovered that one of these, the type VI secretion system, uses a contractile tube to puncture adjacent cells and inject toxins and other molecules. These tubes turn out to be structural homologs of phage tails, generating interesting hypotheses about their evolutionary relationships.
Cell division. At least three fundamentally different protein machines are known to drive cell division in different organisms: the FtsZ-based bacterial "divisome," the ESCRT system in archaea and eukarya, and actinomysin rings. Our images of the FtsZ and ESCRT machines in their native states within dividing cells have revealed key aspects of how each drives constriction.
Structural Biology of HIV
In addition to imaging small cells, we are also investigating the structural biology of the human immunodeficiency virus type 1 (HIV-1). HIV-1 is unusual: although each virus has the same basic membrane and protein layers, viral particles are all unique, making standard methods such as x-ray crystallography or EM-based single-particle analysis ineffective. Thus, while there are already hundreds of structures of individual HIV protein domains available in the Protein Data Bank, many questions remain about how these assemble to form a virus and how subsequent protein modifications drive maturation. We have used cryotomography to determine the 3D structures of the immature and mature forms of HIV-1, and we are now working to understand transient states involved in budding, maturation, and uncoating.
Electron cryomicroscopy is a relatively young field, and we are working to improve sample preparation methods, the number and quality of images produced, and methods to process and analyze those images. We have, for example, developed tools and protocols for correlated light and electron microscopies, described a new cryogen mixture that facilitates plunge-freezing, shown that liquid nitrogen is a better coolant than liquid helium, characterized the first cryo-stage for routine dual-axis tilting, begun testing a new direct electron detector, automated sequential tilt-series acquisition, developed a model database for storing tomograms, and developed an automated pipeline for processing images.
This work is also supported by the National Institutes of Health, the Beckman Institute at Caltech, the Agouron Institute, and the Gordon and Betty Moore Foundation.
As of August 30, 2012