Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution ...microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
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•Stress granules are composed of stable cores surrounded by a phase-separated shell•Purification of cores identified new members of the stress granule proteome•Granule assembly and dynamics are modulated by ATP•CCT, RVB, and MCM ATPases regulate distinct steps in granule assembly/disassembly
Although current models describe stress granules as liquid-like assemblies, super-resolution microscopy reveals that granules contain stable core substructures surrounded by a phase-separated shell.
Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less ...concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.
Electrochemistry, biosensors and microfluidics are popular research topics that have attracted widespread attention from chemists, biologists, physicists, and engineers. Here, we introduce the basic ...concepts and recent histories of electrochemistry, biosensors, and microfluidics, and describe how they are combining to form new application-areas, including so-called "point-of-care" systems in which measurements traditionally performed in a laboratory are moved into the field. We propose that this review can serve both as a useful starting-point for researchers who are new to these topics, as well as being a compendium of the current state-of-the art for experts in these evolving areas.
Crop production is inherently sensitive to variability in climate. Temperature is a major determinant of the rate of plant development and, under climate change, warmer temperatures that shorten ...development stages of determinate crops will most probably reduce the yield of a given variety. Earlier crop flowering and maturity have been observed and documented in recent decades, and these are often associated with warmer (spring) temperatures. However, farm management practices have also changed and the attribution of observed changes in phenology to climate change per se is difficult. Increases in atmospheric CO₂ often advance the time of flowering by a few days, but measurements in FACE (free air CO₂ enrichment) field-based experiments suggest that elevated CO₂ has little or no effect on the rate of development other than small advances in development associated with a warmer canopy temperature. The rate of development (inverse of the duration from sowing to flowering) is largely determined by responses to temperature and photoperiod, and the effects of temperature and of photoperiod at optimum and suboptimum temperatures can be quantified and predicted. However, responses to temperature, and more particularly photoperiod, at supraoptimal temperature are not well understood. Analysis of a comprehensive data set of time to tassel initiation in maize (Zea mays) with a wide range of photoperiods above and below the optimum suggests that photoperiod modulates the negative effects of temperature above the optimum. A simulation analysis of the effects of prescribed increases in temperature (0-6 °C in +1 °C steps) and temperature variability (0% and +50%) on days to tassel initiation showed that tassel initiation occurs later, and variability was increased, as the temperature exceeds the optimum in models both with and without photoperiod sensitivity. However, the inclusion of photoperiod sensitivity above the optimum temperature resulted in a higher apparent optimum temperature and less variability in the time of tassel initiation. Given the importance of changes in plant development for crop yield under climate change, the effects of photoperiod and temperature on development rates above the optimum temperature clearly merit further research, and some of the knowledge gaps are identified herein.
The digital revolution has come to microfluidics. In digital microfluidics (DMF), discrete droplets are manipulated by applying electrical fields to an array of electrodes. In contrast to ...microchannels, in DMF each sample and reagent is individually addressable, which facilitates exquisite control over chemical reactions. Here, we review the state‐of‐the‐art in DMF, with a discussion of device formats, actuation physics, and biological and nonbiological applications. Along the way, we identify the key players in the field, and speculate on the advances and challenges that lie ahead. As with other fronts in the digital revolution, there have been and will be unexpected developments as DMF matures, but we posit that the future is bright for this promising technology.
We review the state‐of‐the‐art in digital microfluidics, a technique in which discrete droplets are manipulated by applying electrical fields to an array of electrodes. A discussion of device formats, actuation physics, and biological and nonbiological applications is presented. Along the way, we identify the key players in the field, and speculate on the advances and challenges that lie ahead.
Most electroanalytical techniques require the precise control of the potentials in an electrochemical cell using a potentiostat. Commercial potentiostats function as "black boxes," giving limited ...information about their circuitry and behaviour which can make development of new measurement techniques and integration with other instruments challenging. Recently, a number of lab-built potentiostats have emerged with various design goals including low manufacturing cost and field-portability, but notably lacking is an accessible potentiostat designed for general lab use, focusing on measurement quality combined with ease of use and versatility. To fill this gap, we introduce DStat (http://microfluidics.utoronto.ca/dstat), an open-source, general-purpose potentiostat for use alone or integrated with other instruments. DStat offers picoampere current measurement capabilities, a compact USB-powered design, and user-friendly cross-platform software. DStat is easy and inexpensive to build, may be modified freely, and achieves good performance at low current levels not accessible to other lab-built instruments. In head-to-head tests, DStat's voltammetric measurements are much more sensitive than those of "CheapStat" (a popular open-source potentiostat described previously), and are comparable to those of a compact commercial "black box" potentiostat. Likewise, in head-to-head tests, DStat's potentiometric precision is similar to that of a commercial pH meter. Most importantly, the versatility of DStat was demonstrated through integration with the open-source DropBot digital microfluidics platform. In sum, we propose that DStat is a valuable contribution to the "open source" movement in analytical science, which is allowing users to adapt their tools to their experiments rather than alter their experiments to be compatible with their tools.
Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. The morphology of the reproductive organs ...and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes. Plants were exposed to 6 h of high (38 °C) and control (29 °C) temperature at anthesis and spikelets collected for morphological and proteomic analysis. Moroberekan was the most heat-sensitive genotype (18% spikelet fertility at 38 °C), while IR64 (48%) and N22 (71%) were moderately and highly heat tolerant, respectively. There were significant differences among the genotypes in anther length and width, apical and basal pore lengths, apical pore area, and stigma and pistil length. Temperature also affected some of these traits, increasing anther pore size and reducing stigma length. Nonetheless, variation in the number of pollen on the stigma could not be related to measured morphological traits. Variation in spikelet fertility was highly correlated (r=0.97, n=6) with the proportion of spikelets with ≥20 germinated pollen grains on the stigma. A 2D-gel electrophoresis showed 46 protein spots changing in abundance, of which 13 differentially expressed protein spots were analysed by MS/MALDI-TOF. A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22. The role of differentially expressed proteins and morphology during anther dehiscence and pollination in shaping heat tolerance and susceptibility is discussed.
•Stress granule cores are stable ex vivo.•Stress granule cores contain known stress granule components and RNA.•Stress granule cores are purified using a combination of centrifugation followed by ...immunoprecipitation.•A purified population of stress granule cores can be used for biochemical, proteomic, and transcriptomic experiments.
Stress granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian stress granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of stress granule cores from both yeast and mammalian cells. The protocol consists of first enriching for stress granule cores using centrifugation and then further purifying stress granule cores using immunoprecipitation. The stress granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.
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