In Vitro Motility of Actin Filaments Powered by Plant Myosins XI

The actomyosin network is thought to support fundamental processes of plant development and cell expansion such as polarized elongation of root hairs and the diffuse growth of epidermal and mesophyll cells. Inhibition of myosins via pharmacological treatments represents one of the key approaches for understanding of their roles in different cellular processes. However, the use of the standard plant myosin inhibitor, 2,3-butanedionemonoxime (BDM), is questioned as it requires a high concentration and may not be as specific as desired. By testing drugs that inhibit animal and yeast myosins V, the Staiger laboratory previously found pentabromopseudilin (PBP) as a potential inhibitor of plant myosins in vivo. In order to verify PBP as a plant myosin inhibitor in vitro, an actin filament gliding assay powered by chicken Myosin Va (MyoVa) was developed as a positive control using Total Internal Reflection Fluorescence Microscopy (TIRFM). Here, we partially purified a YFP-tagged Myosin XIK from Arabidopsis thaliana, and enriched it in the motility assay chamber by an antibody affinity-capture method. The enriched XIK-YFP showed actin binding activity and addition of ATP resulted in detachment of actin filaments (F-actin) from the protein, suggesting that the ATPase domain of the isolated myosin is partially functional. By testing the detachment frequency of myosin-bound F-actin, we demonstrated that PBP could effectively inhibit the ATP-dependent release of F-actin from the isolated XIK-YFP, suggesting that PBP is a potential plant myosin inhibitor

HYBRID MEMBRANE-PSA SYSTEM FOR SEPARATING OXYGEN FROMAR

A portable, non-cryogenic, oxygen generation system capable of delivering oxygen gas at purities greater than 98% and flow rates of 15 L/min or more is described. The system consists of two major components. The first component is a high efficiency membrane capable of separating argon and a portion of the nitrogen content from air, yielding an oxygen-enriched permeate flow. This is then fed to the second component, a pressure swing adsorption (PSA) unit utilizing a commercially available, but specifically formulated zeolite compound to remove the remainder of the nitrogen from the flow. The system is a unique gas separation system that can operate at ambient temperatures, for producing high purity oxygen for various applications (medical, refining, chemical production, enhanced combustion, fuel cells, etc...) and represents a significant advance compared to current technologies

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3–like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI

Capping protein modulates the dynamic behavior of actin filaments in response to phosphatidic Acid in Arabidopsis.

International audienceRemodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics

The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria

Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly

Mitochondrial Fusion Is Increased by the Nuclear Coactivator PGC-1β

Background There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression. Methodology/Principal Findings Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1β is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1β increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1β-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1β increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor α (ERRα). Conclusions/Significance Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1β in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2

US public opinion regarding proposed limits on resident physician work hours

<p>Abstract</p> <p>Background</p> <p>In both Europe and the US, resident physician work hour reduction has been a source of controversy within academic medicine. In 2008, the Institute of Medicine (IOM) recommended a reduction in resident physician work hours. We sought to assess the American public perspective on this issue.</p> <p>Methods</p> <p>We conducted a national survey of 1,200 representative members of the public via random digit telephone dialing in order to describe US public opinion on resident physician work hour regulation, particularly with reference to the IOM recommendations.</p> <p>Results</p> <p>Respondents estimated that resident physicians currently work 12.9-h shifts (95% CI 12.5 to 13.3 h) and 58.3-h work weeks (95% CI 57.3 to 59.3 h). They believed the maximum shift duration should be 10.9 h (95% CI 10.6 to 11.3 h) and the maximum work week should be 50 h (95% CI 49.4 to 50.8 h), with 1% approving of shifts lasting >24 h (95% CI 0.6% to 2%). A total of 81% (95% CI 79% to 84%) believed reducing resident physician work hours would be very or somewhat effective in reducing medical errors, and 68% (95% CI 65% to 71%) favored the IOM proposal that resident physicians not work more than 16 h over an alternative IOM proposal permitting 30-h shifts with ≥5 h protected sleep time. In all, 81% believed patients should be informed if a treating resident physician had been working for >24 h and 80% (95% CI 78% to 83%) would then want a different doctor.</p> <p>Conclusions</p> <p>The American public overwhelmingly favors discontinuation of the 30-h shifts without protected sleep routinely worked by US resident physicians and strongly supports implementation of restrictions on resident physician work hours that are as strict, or stricter, than those proposed by the IOM. Strong support exists to restrict resident physicians' work to 16 or fewer consecutive hours, similar to current limits in New Zealand, the UK and the rest of Europe.</p