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FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins 总被引:1,自引:1,他引:1 下载免费PDF全文
Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae. 相似文献
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Goudarzi M Banisch TU Mobin MB Maghelli N Tarbashevich K Strate I van den Berg J Blaser H Bandemer S Paluch E Bakkers J Tolić-Nørrelykke IM Raz E 《Developmental cell》2012,23(1):210-218
Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in?vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which confers proper morphology and cell motility. Zebrafish primordial germ cells rendered immotile by knockdown of Dead end, a negative regulator of miRNA function, were used as a platform for identifying processes restoring motility. We have defined myosin contractility, cell adhesion, and cortex properties as factors whose proper regulation is sufficient for restoring cell migration of this cell type. Tight control over the level of these cellular features, achieved through a balance between miRNA-430 function and the action of the RNA-binding protein Dead end, effectively transforms immotile primordial germ cells into polarized cells that actively migrate relative to cells in their environment. 相似文献
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Boldajipour B Doitsidou M Tarbashevich K Laguri C Yu SR Ries J Dumstrei K Thelen S Dörries J Messerschmidt EM Thelen M Schwille P Brand M Lortat-Jacob H Raz E 《Development (Cambridge, England)》2011,138(14):2909-2914
The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes. 相似文献
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Zimyanin VL Belaya K Pecreaux J Gilchrist MJ Clark A Davis I St Johnston D 《Cell》2008,134(5):843-853
oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport. 相似文献
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Arifa Sosan Dimitri Svistunenko Darya Straltsova Katsiaryna Tsiurkina Igor Smolich Tracy Lawson Sunitha Subramaniam Vladimir Golovko David Anderson Anatoliy Sokolik Ian Colbeck Vadim Demidchik 《The Plant journal : for cell and molecular biology》2016,85(2):245-257
Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L?1, inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca2+]cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole‐cell patch‐clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K+ efflux and Ca2+ influx currents, or caused membrane breakdown; however, in excised outside‐out patches, Ag NPs activated Gd3+‐sensitive Ca2+ influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l ‐ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca2+]cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate. 相似文献
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Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos. 相似文献
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Dhiraj?Sinha Vitali?Bialevich Katsiaryna?Shamayeva Alena?Guzanova Alexandra?Sisakova Eva?Csefalvay David?Reha Lumir?Krejci Jannette?Carey Marie?Weiserova Rüdiger?EttrichEmail authorView authors OrcID profile 《Journal of molecular modeling》2018,24(7):176
Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains. 相似文献
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Thomas Palm Kathrin Hemmer Julia Winter Inga B. Fricke Katsiaryna Tarbashevich Fereshteh Sadeghi Shakib Ina-Maria Rudolph Anna-Lena Hillje Paola De Luca Lamia'a Bahnassawy Rabea Madel Thomas Viel Adriana De Siervi Andreas H. Jacobs Sven Diederichs Jens C. Schwamborn 《Nucleic acids research》2013,41(6):3699-3712