Search

This study of an upwelling cross-shore filament in the California Current System attempts to follow a parcel of water from upwelling source to offshore in order to characterize changes in silica cycling and associated impacts on carbon cycling under the influence of iron limitation. Water column samples were collected throughout the euphotic zone in conjunction with sediment traps following the filament in Lagrangian fashion (cycles); additional samples were taken across the filament (transects). The filament progressed from iron-replete to iron-limited throughout the study, identified by low silicic acid to nitrate ratios (Si:N) and elevated nitrate to iron (N:Fe) ratios in situ. Increases in biogenic silica to POC ratios (bSi:POC) in the water column were apparent with increasing iron limitation of the diatom-dominated community. The Lagrangian nature of the study improves overall understanding of the dynamics of biogenic silica as an upwelling filament develops to iron-limited, low Si:N water conditions. This work builds upon and adds context to previous studies which have shown that iron limitation affects silicic acid to carbon (Si:C) and silicic acid to nitrate (Si:N) uptake ratios in marine diatoms. These ratios have potential implications for both carbon and silica cycling in the ocean, impacting uptake and export of these elements. The filament was shown to transport biogenic silica laterally away from the coast, and enhanced silica ballasting was shown to be associated with increased export efficiency offshore.

Plasma blobs (filamentary structures extended along the magnetic field lines) are mesoscale turbulent structures that are usually observed in the scrape-off layer (SOL) of magnetic confinement devices. Electromagnetic fluid simulations for seeded high-beta blobs demonstrate that inhomogeneity of magnetic curvature or plasma pressure along the filament leads to bending of the filaments and magnetic field lines due to increased Alfven time. Moreover, the drift wave instabilities inside the filaments are modified by electromagnetic effects. A local linear analysis on an electromagnetic drift-kinetic equation with BGK-like collision operator proves to be valid for describing the linear growth rate of drift wave instabilities in a wide range of plasma parameters showing convergence to reference models for limiting cases. Finally, the development of the 5D version of the full-f gyrokinetic code COGENT and its application to the collisionless drift wave simulation are presented with discussion of the disturbance of the distribution function from the collisionless drift wave instability and the formation of a helical mode structure within the plasma filament.

Tubulin is a universally conserved molecule, found in all three domains of life. Tubulin filaments use energy derived from GTP binding and hydrolysis to organize cytoplasm. Although tubulin homologues share the same fold to their monomers and similar protofilament architecture, they assemble filaments with unique geometric designs. Moreover, despite sharing the mechanism of self-assembly, wherein tubulin subunits require GTP binding to polymerize, and their polymerization stimulates GTP hydrolysis, tubulins exhibit distinctive dynamic properties. Together, unique filament architectures and dynamic properties determine a specific set of biological functions each tubulin family performs. The origins of tubulin filament architectural diversity and distinctive dynamic behavior are not well understood. We have been studying PhuZ tubulins, which are encoded by a few very large Pseudomonas ϕKZ-like bacteriophages. Our studies have shown that PhuZ assembles dynamically unstable spindle-like arrays that organize bacteriophage DNA at the cell midpoint, which somehow facilitates phage infectivity. Moreover, PhuZ monomer structure has the canonical tubulin fold, with a unique, highly conserved and extended C-terminus. The C-terminus mediates protofilament contacts and is critical for polymerization both in vitro and in vivo. The main focus of this manuscript is the structural investigation of the molecular mechanisms underlying PhuZ dynamic behavior. In an attempt to explain how GTP binding and hydrolysis drives PhuZ filament turnover, we have conducted a number of electron cryomicroscopy studies (cryo-EM) on PhuZ filaments in both pre- and post- hydrolysis states. PhuZ forms a polar three-stranded polymer with an unusual subunit orientation. Its C-terminus guides a cooperative assembly of the three-stranded filament by mediating both longitudinal and lateral interactions. Upon assembly, the longitudinal interface, C-terminus and tubulin fold undergo polymerization-competent rearrangements. We propose that the energy of GTP binding is stored in the displacement of these structural elements. Also, our structural studies have revealed how the energy of GTP hydrolysis is stored in the PhuZ filament lattice. In particular, GTP hydrolysis, initially sensed by the T3 loop and C-terminus, is accompanied by unwinding and supercoiling of the filament lattice. Based on these observations, we propose an assembly/disassembly pathway of PhuZ filament.

The tubulin superfamily is a unique set of GTPases, which, while they have high sequence diversity, have remarkably similar tertiary structures. These proteins share a very conserved longitudinal interface, but are able to form a surprisingly wide variety of super structures within both eukaryotic and prokaryotic cells — from the familiar 13-protofilament microtubule assembled from αβ–tubulin heterodimers to the single protofilaments of the ubiquitous prokaryotic FtsZ seen in vitro. Additionally in eukaryotes, monomeric tubulin family members have the ability to affect the assembly of filaments, such as nucleation of microtubules with γ–tubulin, or involvement in the assembly of B– and C– microtubules in the centriole microtubule triplets (ε– and δ–tubulin respectively). Each tubulin family member, both prokaryotic and eukaryotic is exquisitely tuned to perform its cellular function, with a unique superstructure and dynamics. The main focus of this manuscript is a structural and biochemical investigation of a recently discovered bacterial tubulin TubZ–Bt. TubZ–Bt is located on the pBtoxis virulence plasmid in Bacillus thuringiensis, and is necessary to maintain stability of the pBtoxis plasmid in conjunction with an upstream DNA–adaptor protein (TubR) and DNAbinding site (tubC). We have determined that TubZ–Bt forms four–stranded filaments in the presence of GTP in vitro, whereas TubZ filaments formed with catalytically compromised protein or non-hydrolysable GTP analogs are predominantly two-stranded. These morphological changes were investigated by high–resolution cryo–electron microscopy of both two– and four–stranded TubZ–Bt filaments, as well as various biochemical approaches, and we propose that TubZ–Bt two–stranded filaments that we observed are an intermediate on pathway to four–stranded filament formation. In addition, we have begun preliminary investigations on the interaction of TubR bind to tubC DNA and the interaction of the TubRC complex with TubZ-Bt filaments.A minor focus of this manuscript is work on the eukaryotic tubulins, γ–, δ– and ε–tubulin. This work details preliminary attempts at purification of these tubulin family members in order to begin more detailed investigative work on their molecular function. Also included is a published manuscript of work done with Dr. Luke Rice investigating the conformational state of both αβ– and γ–tubulins using solution–state and crystallographic structural studies.

© 2018 by the authors. Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin bundles, traditional approaches to filament detection and tracing have failed in these cases. In this study, we introduce BundleTrac, an effective method to trace hundreds of filaments in a bundle. A comparison between BundleTrac and manually tracing the actin filaments in a stereocilium showed that BundleTrac accurately built 326 of 330 filaments (98.8%), with an overall cross-distance of 1.3 voxels for the 330 filaments. BundleTrac is an effective semi-automatic modeling approach in which a seed point is provided for each filament and the rest of the filament is computationally identified. We also demonstrate the potential of a denoising method that uses a polynomial regression to address the resolution and high-noise anisotropic environment of the density map.