Tubulin isoform composition tunes microtubule dynamics

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2Å cryo-EM structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared to brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Lastly, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step towards understanding how tubulin isoform composition tunes microtubule dynamics

First Precambrian palaeomagnetic data from the Mawson Craton (East Antarctica) and tectonic implications

A pilot palaeomagnetic study was conducted on the recently dated with in situ SHRIMP U-Pb method at 1134 ± 9 Ma (U-Pb, zircon and baddeleyite) Bunger Hills dykes of the Mawson Craton (East Antarctica). Of the six dykes sampled, three revealed meaningful results providing the first well-dated Mesoproterozoic palaeopole at 40.5°S, 150.1°E (A95 = 20°) for the Mawson Craton. Discordance between this new pole and two roughly coeval poles from Dronning Maud Land and Coats Land (East Antarctica) demonstrates that these two terranes were not rigidly connected to the Mawson Craton ca. 1134 Ma. Comparison between the new pole and that of the broadly coeval Lakeview dolerite from the North Australian Craton supports the putative ~40° late Neoproterozoic relative rotation between the North Australian Craton and the combined South and West Australian cratons. A mean ca. 1134 Ma pole for the Proto-Australia Craton is calculated by combining our new pole and that of the Lakeview dolerite after restoring the 40° intracontinental rotation. A comparison of this mean pole with the roughly coeval Abitibi dykes pole from Laurentia confirms that the SWEAT reconstruction of Australia and Laurentia was not viable for ca. 1134 Ma

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target

Atypical Balance between Occipital and Fronto-Parietal Activation for Visual Shape Extraction in Dyslexia

Reading requires the extraction of letter shapes from a complex background of text, and an impairment in visual shape extraction would cause difficulty in reading. To investigate the neural mechanisms of visual shape extraction in dyslexia, we used functional magnetic resonance imaging (fMRI) to examine brain activation while adults with or without dyslexia responded to the change of an arrow’s direction in a complex, relative to a simple, visual background. In comparison to adults with typical reading ability, adults with dyslexia exhibited opposite patterns of atypical activation: decreased activation in occipital visual areas associated with visual perception, and increased activation in frontal and parietal regions associated with visual attention. These findings indicate that dyslexia involves atypical brain organization for fundamental processes of visual shape extraction even when reading is not involved. Overengagement in higher-order association cortices, required to compensate for underengagment in lower-order visual cortices, may result in competition for top-down attentional resources helpful for fluent reading.Ellison Medical FoundationMartin Richmond Memorial FundNational Institutes of Health (U.S.). (Grant UL1RR025758)National Institutes of Health (U.S.). (Grant F32EY014750-01)MIT Class of 1976 (Funds for Dyslexia Research

P2 receptor-mediated modulation of neurotransmitter release—an update

Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions

The elegans of spindle assembly

The Caenorhabditis elegans one-cell embryo is a powerful system in which to study microtubule organization because this large cell assembles both meiotic and mitotic spindles within the same cytoplasm over the course of 1 h in a stereotypical manner. The fertilized oocyte assembles two consecutive acentrosomal meiotic spindles that function to reduce the replicated maternal diploid set of chromosomes to a single-copy haploid set. The resulting maternal DNA then unites with the paternal DNA to form a zygotic diploid complement, around which a centrosome-based mitotic spindle forms. The early C. elegans embryo is amenable to live-cell imaging and electron tomography, permitting a detailed structural comparison of the meiotic and mitotic modes of spindle assembly

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700