On a cool afternoon in late March in Bristol, the biannual symposium i-Docs 2018 kicks off with the three co-directors Judith Aston, Sandra Gaudenzi and Mandy Rose riffing on the ‘i's’ or eyes or a...
Abstract Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal αβ-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of Ca Kip3 folds into a compact 92 Å-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.
Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide β-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.
The authors reflect on creating a collaborative creative work that was developed both with, and as, a manifesto. Using queer theory as a framework, the authors track the process of developing and deploying a 14-step manifesto and outline their aims for queering screen production through creative practice. The project applies Baker's (2011) call for a queer-ing of practice-led research, enacting a performative bricolage with a focus on queer screen production that is concerned with more than representation. The resulting 14-minute assemblage film outlines its thesis within an experimental, non-linear structure, comprising clips from the individual authors' previously produced screen works, interplayed with new content, personal archive and textual elements. It combines the authors' separate practices in filmmaking, screenwriting, mobile media and documentary in ways that deviate from mainstream categorisations, production hierarchies and workflows. Firstly, the manifesto is situated among others that outline strategies of disruption and resistance. Then, framed by the manifesto steps, the authors reflect on the film's disruption of dominant narrative models in the context of queer theory's critiques of heteronormative temporality. They then draw some conclusions around the possibilities of 'manifesto as method', and the implications for narrative disruption, queer screen production, and creative practice more broadly.
ABSTRACT This study employed multiple regression analysis to examine the relationship between global writing quality (holistic scores) and lower level analytic measures of writing, with a focus on cohesive indices. The subjects were 9-year-old English-speaking children who participated in either a story free-writing condition or a story rewriting condition. The results showed that both cohesive indices and lower level writing measures (type-token ratios, mean length of utterances in morphemes, composition length, etc.) each accounted for a significant amount of the variance in holistic scores. The story rewriting procedure proved to facilitate the children's writing processes and, hence, resulted in higher quality writing (in terms of both global writing quality and text cohesion) than the story free-writing condition.
Surface plasmon resonance (SPR)-based biosensing is a powerful tool to study the recognition processes between biomolecules in real-time without need for labels. The use of thiol chemistry is a critical component in surface functionalization of various SPR biosensor surfaces on gold. However, its use is hampered by the high propensity for oxidation of the gold-thiol linkage even in ambient atmosphere, resulting in a short lifetime of SPR sensor chips unless strict precautions are taken. Herein, we describe an approach to overcome this limitation by employing highly robust self-assembled monolayers (SAMs) of alkylated N-heterocyclic carbenes (NHCs) on gold. An alkylated NHC sensor surface was developed and its biosensing capabilities were compared to a commercial thiol-based analogue-a hydrophobic association (HPA) chip-in terms of its ability to act as a reliable platform for biospecific interaction analysis under a wide range of conditions. The NHC-based SPR sensor outperforms related thiol-based sesnsors in several aspects, including lower nonspecific binding capacity, better chemical stability, higher reproducibility, shorter equilibration time, and longer life span. We also demonstrate that the NHC-based sensor can be used for rapid and efficient formation of a hybrid lipid bilayer for use in membrane interaction studies. Overall, this work identifies the great promise in designing NHC-based surfaces as a new technology platform for SPR-based biosensing.
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Documentary has traditionally been used and understood as a communicative tool to frame and impart knowledge about a subject matter. For Paula Rabinowitz, documentary’s “purpose is to speak and confer value on the objects it speaks about” (1994). But what other functions might documentary have if it were to draw attention to how we can also listen? Over the past twenty-five year, listening scholarship has focused on a number of domains including the public sphere (Lacey 2013), participatory democracy (Bickford 1996; Couldry 2010), and media practices (Dreher 2009). Yet, while ‘voice’ as authorship and social participation has been well theorised in documentary, there has been little scholarly attention given to how audiences listen, and what strategies creators can use to evoke different ways of listening. The possibilities for listening as an active role in documentary have further been enhanced through interactive technologies. In this presentation, I draw on my current research and practice around the possibilities for interactive and expanded documentary to promote listening as a way to attend to ethical and political questions. Listening is not a singular action, rather, it can fulfill multiple functions. I propose that practices of listening can reframe the self as collective, reveal multiple co-existing narratives, counter erasure, promote engagement with difference and destabilise a anthropocentric perspective. Drawing on frameworks which include political, non-representational and sound theory, I ground my propositions through a range of interactive and immersive documentaries from the past ten years, each of which attempts to implicate the audience in new and responsible relationships to the historical and phenomenological world.
Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca(2+) signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca(2+)-dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa.
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