Septins are a family of GTP-binding, membrane-interacting cytoskeletal proteins, highly conserved and essential in all eukaryotes (with the exception of plants). Septins play important roles in a number of cellular events that involve membrane remodeling and compartmentalization. One such event is cytokinesis, the last stage of cell division. While cytokinesis is ultimately achieved via the mechanical contraction of an actomyosin ring at the septum, determination of the location where cytokinesis will take place, and recruitment of factors involved in signaling events leading to septation requires the activity of septins. We are working towards dissecting the properties of septins from the budding yeast Saccharomyces cerevisiae, where they were first discovered as cell cycle mutants. In our studies we have employed several complementary electron microscopy techniques to describe the organization and structure of septins both in vitro and in situ.

Septins are essential for the completion of cytokinesis. In budding yeast, Saccharomyces cerevisiae, septins are located at the bud neck during mitosis and are closely connected to the inner plasma membrane. In vitro, yeast septins have been shown to self-assemble into a variety of filamentous structures, including rods, paired filaments, bundles, and rings (Bertin et al. Proc Natl Acad Sci U S A, 105(24):8274-8279, 2008; Garcia et al. J Cell Biol, 195(6):993-1004, 2011; Bertin et al. J Mol Biol, 404(4):711-731, 2010). Using electron tomography of freeze-substituted sections and cryo-electron tomography of frozen sections, we determined the three-dimensional organization of the septin cytoskeleton in dividing budding yeast with molecular resolution (Bertin et al. Mol Biol Cell, 23(3):423-432, 2012; Bertin and Nogales. Commun Integr Biol 5(5):503-505, 2012). Here, we describe the detailed procedures used for our characterization of the septin cellular ultrastructure.

Septins are highly conserved and essential eukaryotic cytoskeletal proteins that interact with the inner plasma membrane. They are involved in essential functions requiring cell membrane remodeling and compartmentalization, such as cell division and dendrite morphogenesis, and have been implicated in numerous diseases. Depending on the organisms and on the type of tissue, a specific set of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into linear, symmetric rods that can further organize into linear filaments several microns in length. Only a subset of human septins has been described at high resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron microscopy (EM) has proven to be a method of choice for analyzing the molecular organization of septins. It is possible to localize each septin subunit within the rod complex using genetic tags, such as maltose-binding protein or green fluorescent protein, to generate a visible label of a specific septin subunit in EM images that are processed using single-particle EM methodology. In this chapter we present, in detail, the methods that we have used to analyze the molecular organization of budding yeast septins (Bertin et al., 2008). These methods include purification of septin complexes, sample preparation for EM, and image processing procedures. Such methods can be generalized to analyze the organization of septins from any organism.

When a material is plastically deformed the majority of mechanical work is dissipated as heat, and the fraction of plastic work converted into heat is known as the Taylor-Quinney coefficient (TQC). Large-scale molecular dynamics simulations were performed of high strain rate compression of single-crystal tantalum, and the resulting integral and differential TQC values are reported up to true strains of 1.0. A phenomenological model is proposed for the energy stored in the material as a function of plastic strain with an asymptotic limit for this energy defined by the deformation conditions. The model reasonably describes the convergence of TQC values to 1.0 with increasing plastic strain, but does not directly address the physical nature of thermo-mechanical conversion. This is instead developed in a second more detailed model that accurately accounts for energy storage with two distinct contributions, one being the growing dislocation network and the other the point defect debris left behind by moving dislocations. The contribution of the point defect debris is found to lag behind that of the dislocation network but to be substantial for the high-rate straining conditions considered here.

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.

Septins are conserved guanosine triphosphate-binding cytoskeletal proteins involved in membrane remodeling. In budding yeast, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1), which are essential for cytokinesis, transition during bud growth from a patch to a collar, which splits into two rings in cytokinesis and is disassembled before the next cell cycle. Cdc3, Cdc10, Cdc11, and Cdc12 form an apolar octameric rod with Cdc11 at each tip, which polymerizes into straight paired filaments. We show that Shs1 substitutes for Cdc11, resulting in octameric rods that do not polymerize into filaments but associate laterally, forming curved bundles that close into rings. In vivo, half of shs1Δ mutant cells exhibit incomplete collars and disrupted neck filaments. Importantly, different phosphomimetic mutations in Shs1 can either prevent ring formation or promote formation of a gauzelike meshwork. These results show that a single alternative terminal subunit is sufficient to confer a distinctive higher-order septin ultrastructure that can be further regulated by phosphorylation.

Understanding of the capacity of the natural world to produce secondary metabolites is important to a broad range of fields, including drug discovery, ecology, biosynthesis, and chemical biology, among others. Both the absolute number and the rate of discovery of natural products have increased significantly in recent years. However, there is a perception and concern that the fundamental novelty of these discoveries is decreasing relative to previously known natural products. This study presents a quantitative examination of the field from the perspective of both number of compounds and compound novelty using a dataset of all published microbial and marine-derived natural products. This analysis aimed to explore a number of key questions, such as how the rate of discovery of new natural products has changed over the past decades, how the average natural product structural novelty has changed as a function of time, whether exploring novel taxonomic space affords an advantage in terms of novel compound discovery, and whether it is possible to estimate how close we are to having described all of the chemical space covered by natural products. Our analyses demonstrate that most natural products being published today bear structural similarity to previously published compounds, and that the range of scaffolds readily accessible from nature is limited. However, the analysis also shows that the field continues to discover appreciable numbers of natural products with no structural precedent. Together, these results suggest that the development of innovative discovery methods will continue to yield compounds with unique structural and biological properties.

Earth’s magnetic field is generated by convective motions in its liquid metal core. In this fluid, the heat diffuses significantly more than momentum, and thus the Prandtl number $Pr$ is well below unity. The thermally driven convective flow dynamics of liquid metals are very different from moderate-$Pr$ fluids, such as water and those used in current dynamo simulations. In order to characterise rapidly rotating thermal convection in low-$Pr$ number fluids, we have performed laboratory experiments in a cylinder of aspect ratio $\unicode[STIX]{x1D6E4}=1.94$ using liquid gallium ($Pr\simeq 0.025$) as the working fluid. The Ekman number varies from $E\simeq 5\times 10^{-6}$ to $5\times 10^{-5}$ and the Rayleigh number varies from $Ra\simeq 2\times 10^{5}$ to $1.5\times 10^{7}$. Using spectral analysis stemming from point-wise temperature measurements within the fluid and measurements of the Nusselt number $Nu$, we characterise the different styles of low-$Pr$ rotating convective flow. The convection threshold is first overcome in the form of container-scale inertial oscillatory modes. At stronger forcing, sidewall-attached modes are identified for the first time in liquid metal laboratory experiments. These wall modes coexist with the bulk oscillatory modes. At $Ra$ well below the values where steady rotating columnar convection occurs, the bulk flow becomes turbulent. Our results imply that rotating convective flows in liquid metals do not develop in the form of quasisteady columns, as in moderate-$Pr$ fluids, but in the form of oscillatory convective motions. Thus, thermally driven flows in low-$Pr$ geophysical and astrophysical fluids can differ substantively from those occurring in $Pr\simeq 1$ models. Furthermore, our experimental results show that relatively low-frequency wall modes are an essential dynamical component of rapidly rotating convection in liquid metals.

Members of the cyanobacterial genus Trichodesmium are well known for their substantial impact on nitrogen influx in ocean ecosystems and the enormous surface blooms they form in tropical and subtropical locations. However, the secondary metabolite composition of these complex environmental bloom events is not well known, nor the possibility of the production of potent toxins that have been observed in other bloom-forming marine and freshwater cyanobacteria species. In the present work, we aimed to characterize the metabolome of a Trichodesmium bloom utilizing MS/MS-based molecular networking. Furthermore, we integrated cytotoxicity assays in order to identify and ultimately isolate potential cyanotoxins from the bloom. These efforts led to the isolation and identification of several members of the smenamide family, including three new smenamide analogs (1-3) as well as the previously reported smenothiazole A-hybrid polyketide-peptide compounds. Two of these new smenamides possessed cytotoxicity to neuro-2A cells (1 and 3) and their presence elicits further questions as to their potential ecological roles. HPLC profiling and molecular networking of chromatography fractions from the bloom revealed an elaborate secondary metabolome, generating hypotheses with respect to the environmental role of these metabolites and the consistency of this chemical composition across genera, space and time.