In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5

Ohrt, Thomas; Merkle, Dennis; Birkenfeld, Karin; Echeverri, Christophe J.; Schwille, Petra

doi:10.1093/nar/gkl001

Cited by 93 publications

(85 citation statements)

References 54 publications

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“…To explore this possibility, we analyzed the effect of EB1 depletion on podosome patterning. We first used siRNAs delivered directly to the cytoplasm via microinjection (39). The functionality of several EB1 siRNAs was first tested and validated in the murine macrophage cell line RAW264.7, transfected either with the targeted siRNA or with a universal negative control.…”

Section: Resultsmentioning

confidence: 99%

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

Duplan

Zalli

Stephens

et al. 2014

Molecular and Cellular Biology

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In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (؉TIPs) might regulate podosome patterning. Among the ؉TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.

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Section: Resultsmentioning

confidence: 99%

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

Duplan

Zalli

Stephens

et al. 2014

Molecular and Cellular Biology

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“…Because Rsp s alleles have many (and Rsp i alleles have few) repeats, Rsp s -bearing spermatids might be disproportionately affected by the dearth of Rsp rasiRNAs in the nucleus and as a result fail to condense their pericentric heterochromatin (Ferree and Barbash 2007;Tao et al 2007). While the Ran cycle is involved in nuclear export of miRNAs (Ohrt et al 2006;Mtango et al 2009), its role in rasiRNA transport is unknown. It is possible then that neither Ran nor ran-like are responsible for the postmeiotic import of rasiRNAs to their target satellites.…”

Section: Segregation Distortion and Small Rnasmentioning

confidence: 99%

The SelfishSegregation DistorterGene Complex ofDrosophila melanogaster

2012

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Segregation Distorter (SD) is an autosomal meiotic drive gene complex found worldwide in natural populations of Drosophila melanogaster. During spermatogenesis, SD induces dysfunction of SD + spermatids so that SD/SD + males sire almost exclusively SD-bearing progeny rather than the expected 1:1 Mendelian ratio. SD is thus evolutionarily "selfish," enhancing its own transmission at the expense of its bearers. Here we review the molecular and evolutionary genetics of SD. Genetic analyses show that the SD is a multilocus gene complex involving two key loci-the driver, Segregation distorter (Sd), and the target of drive, Responder (Rsp)-and at least three upward modifiers of distortion. Molecular analyses show that Sd encodes a truncated duplication of the gene RanGAP, whereas Rsp is a large pericentromeric block of satellite DNA. The Sd-RanGAP protein is enzymatically wild type but mislocalized within cells and, for reasons that remain unclear, appears to disrupt the histone-to-protamine transition in drive-sensitive spermatids bearing many Rsp satellite repeats but not drive-insensitive spermatids bearing few or no Rsp satellite repeats. Evolutionary analyses show that the Sd-RanGAP duplication arose recently within the D. melanogaster lineage, exploiting the preexisting and considerably older Rsp satellite locus. Once established, the SD haplotype collected enhancers of distortion and suppressors of recombination. Further dissection of the molecular genetic and cellular basis of SD-mediated distortion seems likely to provide insights into several important areas currently understudied, including the genetic control of spermatogenesis, the maintenance and evolution of satellite DNAs, the possible roles of small interfering RNAs in the germline, and the molecular population genetics of the interaction of genetic linkage and natural selection.Mendelian inheritance is a marvelous device for making evolution by natural selection an efficient process.... The Mendelian system works with maximum efficiency only if it is scrupulously fair to all genes. It is in constant danger, however, of being upset by genes that subvert the meiotic process to their own advantage. James F. Crow (1979) S EGREGATION Distorter (SD) is a selfish, coadapted gene complex on chromosome 2 (an autosome) found at low frequency in nearly all natural populations of the fruit fly, Drosophila melanogaster. In heterozygous males carrying SD and a typical wild-type second chromosome (SD/SD + ), most SD + -bearing spermatid nuclei fail to complete the histoneto-protamine transition during spermiogenesis, so that primarily SD-bearing spermatids develop properly and go on to fertilize eggs. SD/SD + males thus sire almost exclusively SD-inheriting progeny. This distortion of classic Mendelian ratios has intrigued geneticists and evolutionary biologists for more than 50 years-and for good reason. As we describe below, SD is a newly evolved system that subverts one of the fundamental laws of inheritance by exploiting an ancient molecular pathway. I...

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“…The cross-correlation value, a ratio of the number of the fluorescent complexes containing both proteins of interest to the number of the fluorescent species of one of the proteins, was used to estimate the micelles with more than one BAX molecule. As a positive control and as a calibration sample, the 21-bp-long double-stranded RNA labeled with AlexaFluor 488 and Cy5 on each 3Ј-end was used (25).…”

Section: Calculation Of Protein-detergent Micelle Molecular Weight Bamentioning

confidence: 99%

Detergent-activated BAX Protein Is a Monomer

Ivashyna

García‐Sáez

Ries

et al. 2009

Journal of Biological Chemistry

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BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation withdetergents,suchasn-octylglucoside.DuringBAXactivationwith n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.

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In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5

Cited by 93 publications

References 54 publications

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

The SelfishSegregation DistorterGene Complex ofDrosophila melanogaster

Detergent-activated BAX Protein Is a Monomer

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