Visualizing protein interactions involved in the formation of the 42S RNP storage particle of Xenopus oocytes

2010 ◽  
Vol 102 (8) ◽  
pp. 469-478 ◽  
Author(s):  
Hannah Schneider ◽  
Marie-Christine Dabauvalle ◽  
Norbert Wilken ◽  
Ulrich Scheer
Keyword(s):  
Protein Interactions ◽  
Xenopus Oocytes ◽  
Storage Particle

scholarly journals PTB/hnRNP I Is Required for RNP Remodeling during RNA Localization in Xenopus Oocytes

2007 ◽  
Vol 28 (2) ◽  
pp. 678-686 ◽  
Author(s):  
Raymond A. Lewis ◽  
James A. Gagnon ◽  
Kimberly L. Mowry
Keyword(s):  
Protein Interactions ◽  
Xenopus Oocytes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Rna Transport ◽  
Rna Sequences ◽  
Cytoplasmic Rna ◽  
Embryonic Polarity ◽  
Specific Mrnas

ABSTRACT Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.

scholarly journals ENaC–Membrane Interactions

2004 ◽  
Vol 123 (6) ◽  
pp. 709-727 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Weijian Shao ◽  
Fengli Guo ◽  
Mark Zeidel ◽  
Warren G. Hill
Keyword(s):  
Protein Interactions ◽  
Xenopus Oocytes ◽  
Feedback Inhibition ◽  
Membrane Conductance ◽  
Membrane Lipid ◽  
Na Channel ◽  
Membrane Area ◽  
Temperature Changes ◽  
Lipid Order ◽  
Effects Of Temperature

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459–6463). As these changes of temperature affect membrane lipid order and lipid–protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order–dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2°C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.

Halothane Inhibits Signaling through m1 Muscarinic Receptors Expressed in Xenopus Oocytes 

1995 ◽  
Vol 82 (1) ◽  
pp. 174-182 ◽  
Author(s):  
Marcel E. Durieux
Keyword(s):  
Angiotensin Ii ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Xenopus Oocytes ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Signaling Systems ◽  
M1 Muscarinic

Background Interactions between volatile anesthetics and muscarinic acetylcholine receptors have been studied primarily in binding assays or in functional systems derived from tissues or cells, often containing multiple receptor subtypes. Because interactions with muscarinic signaling systems may explain some effects and side effects of anesthetics and form a model for anesthetic-protein interactions in general, the author studied anesthetic inhibition of muscarinic signaling in an isolated system. Methods mRNA encoding the m1 muscarinic receptor subtype was prepared in vitro and expressed in Xenopus oocytes. Effects of halothane on methylcholine-induced intracellular Ca2+ release was measured. Angiotensin II receptors were expressed to evaluate anesthetic effects on intracellular signaling. Results m1 Receptors expressed in oocytes were functional, and could be inhibited by atropine and pirenzepine. Halothane depressed m1 muscarinic signaling in a dose-dependent manner: half-maximal inhibition of 10(-7) M methylcholine was obtained with 0.3 mM halothane. The effect was reversible and could be overcome by high concentrations of muscarinic agonist. Angiotensin II signaling was unaffected by 0.34 mM halothane. Conclusions m1 Muscarinic signaling is inhibited by halothane, and lack of halothane effect on angiotensin signaling indicates that the intracellular signaling systems of Xenopus oocytes are unaffected. Therefore, the most likely site of halothane action is the receptor and/or G protein. Oocytes provide a versatile system for detailed investigation into the molecular mechanism of anesthetic-protein interactions.

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

Role of small nuclear RNAs in eukaryotic gene expression

2013 ◽  
Vol 54 ◽  
pp. 79-90 ◽  
Author(s):  
Saba Valadkhan ◽  
Lalith S. Gunawardane
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Stability ◽  
Splice Sites ◽  
Branch Site ◽  
Small Nuclear Rnas ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

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