WHITE PAPER: Environmental Scan for DataONE

This environmental scan (conducted by the U&AWG in fall 2018) features a multi-faceted analysis of projects/initiatives in the DataONE space. This report (1) provides context by identifying organizations in the data space; (2) analyzes those organizations most similar to DataONE regarding key services and products; and (3) explores the data training/education environment. As appropriate, the report offers key insights derived from the analysis

An amphipathic helix enables septins to sense micrometer-scale membrane curvature

© The Authors, 2019. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 4.0 International License. The definitive version was published in Journal of Cell Biology (2019), doi:10.1083/jcb.201807211.Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.We thank the Gladfelter laboratory and Danny Lew for useful discussions, Matthias Garten for ideas in setting up the rod assay, and the University of North Carolina EM facility (Victoria Madden and Kristen White) for support with scanning electron microscope. This work was supported by a Howard Hughes Medical Institute Faculty Scholars award to A.S. Gladfelter, and K.S. Cannon was supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999.2019-07-0

The hierarchical assembly of septins revealed by high-speed AFM

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Jiao, F., Cannon, K. S., Lin, Y. C., Gladfelter, A. S., & Scheuring, S. The hierarchical assembly of septins revealed by high-speed AFM. Nature Communications, 11(1), (2020): 5062, doi:10.1038/s41467-020-18778-x.Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.We thank J. Thorner for the generous gift of the CTE mutant plasmids. K.S.C. was supported in part by a grant from NIGMS under award T32 GM119999 and A.S.G., F.J. and S.S. were supported by NIH RO1 GM130934

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Woods, B. L., Cannon, K. S., Vogt, E. J. D., Crutchley, J. M., & Gladfelter, A. S. Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling. Molecular Biology of the Cell, 32(20), (2021): mbcE20050303, https://doi.org/10.1091/mbc.E20-05-0303.The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.This work was supported by National Institutes of Health (NIH) Grant no. R01GM-130934 to A.S.G. B.L.W. was supported by the NIH Training Grant no. 2T32AI052080-16. K.S.C. and E.J.D.V. were supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999

Probing Star Formation at Low Metallicity: The Radio Emission of Super Star Clusters in SBS0335-052

We present high-resolution radio continuum observations of the nascent starburst in the metal-poor galaxy SBS 0335-052. These radio data were taken with the Very Large Array and include observations at 0.7cm, 1.3cm, 2cm, 3.6cm, and 6cm. These observations enable us to probe the thermal radio nebulae associated with the extremely young star-forming regions in this galaxy. Two discrete and luminous star-forming regions are detected in the south of the galaxy that appear to be associated with massive star clusters previously identified at optical wavelengths. However, the remaining optically-identified massive star clusters are not clearly associated with radio emission (either thermal or non-thermal) down to the sensitivity limits of these radio data. The spectral energy distributions of the two radio-detected clusters are consistent with being purely thermal, and the entire region has an inferred ionizing flux of ~1.2 x 10^ 53 s^-1, which is equivalent to ~12,000 "typical" O-type stars (type O7.5 V). The observations presented here have resolved out a significant contribution from diffuse non-thermal emission detected previously, implying a previous episode of significant star formation. The current star formation rate (SFR) for this southern region alone is ~1.3 M_sun yr^-1, or ~ 23M_sun yr^-1 kpc^-2. This SFR derived from thermal radio emission also suggests that previous optical recombination line studies are not detecting a significant fraction of the current star formation in SBS 0335-052. From model fits to the radio spectral energy distribution, we infer a global mean density in the two youngest clusters of n_e > 10^3-10^4 cm^-3. In addition, a comparison between the compact and diffuse radio emission indicates that up to ~50% of the ionizing flux could be leaking out of the compact HII regions.Comment: accepted AJ, 14 pages, 5 figure

Structurally Complex Osteosarcoma Genomes Exhibit Limited Heterogeneity within Individual Tumors and across Evolutionary Time

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNA) are the genetic drivers of disease. Models around genomic instability conflict-it is unclear whether osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single-cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single-cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single-cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little subclonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic timepoints (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. SIGNIFICANCE: Chromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intratumoral heterogeneity and tumor evolution

Optical Measurement Technologies for High Temperature, Radiation Exposure, and Corrosive Environments?Significant Activities and Findings: In-vessel Optical Measurements for Advanced SMRs

Development of advanced Small Modular Reactors (aSMRs) is key to providing the United States with a sustainable, economically viable, and carbon-neutral energy source. The aSMR designs have attractive economic factors that should compensate for the economies of scale that have driven development of large commercial nuclear power plants to date. For example, aSMRs can be manufactured at reduced capital costs in a factory and potentially shorter lead times and then be shipped to a site to provide power away from large grid systems. The integral, self-contained nature of aSMR designs is fundamentally different than conventional reactor designs. Future aSMR deployment will require new instrumentation and control (I&C) architectures to accommodate the integral design and withstand the extreme in-vessel environmental conditions. Operators will depend on sophisticated sensing and machine vision technologies that provide efficient human-machine interface for in-vessel telepresence, telerobotic control, and remote process operations. The future viability of aSMRs is dependent on understanding and overcoming the significant technical challenges involving in-vessel reactor sensing and monitoring under extreme temperatures, pressures, corrosive environments, and radiation fluxe

Assessment, Usability, and Sociocultural Impacts of DataONE

DataONE, funded from 2009-2019 by the U.S. National Science Foundation, is an early example of a large-scale project that built both a cyberinfrastructure and culture of data discovery, sharing, and reuse. DataONE used a Working Group model, where a diverse group of participants collaborated on targeted research and development activities to achieve broader project goals. This article summarizes the work carried out by two of DataONE’s working groups: Usability & Assessment (2009-2019) and Sociocultural Issues (2009-2014). The activities of these working groups provide a unique longitudinal look at how scientists, librarians, and other key stakeholders engaged in convergence research to identify and analyze practices around research data management through the development of boundary objects, an iterative assessment program, and reflection. Members of the working groups disseminated their findings widely in papers, presentations, and datasets, reaching international audiences through publications in 25 different journals and presentations to over 5,000 people at interdisciplinary venues. The working groups helped inform the DataONE cyberinfrastructure and influenced the evolving data management landscape. By studying working groups over time, the paper also presents lessons learned about the working group model for global large-scale projects that bring together participants from multiple disciplines and communities in convergence research