Oxidative DNA damage, particularly 8-oxoguanine (8OG), is a significant contributor to transcriptional errors that can alter the cellular phenotype and cell fate. While previous studies proposed that 8OG can use its a...
R-loops are transcriptionally generated three-stranded nucleic acid structures where the mRNA hybridizes with template DNA, leaving a displaced single-stranded non–template DNA loop. Previously, we demonstrated that ...
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Membrane blebs have important roles in cell migration, apoptosis, and intercellular communication through extracellular vesicles (EVs). While plasma membranes (PM) typically maintain phosphatidylserine (PS) on their c...
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Membrane blebs have important roles in cell migration, apoptosis, and intercellular communication through extracellular vesicles (EVs). While plasma membranes (PM) typically maintain phosphatidylserine (PS) on their cytoplasmic leaflet, most blebs have PS exposed on their outer leaflet, revealing that loss of steady-state lipid asymmetry often accompanies PM blebbing. How these changes in PM lipid organization regulate membrane properties and affect bleb formation remains unknown. We confirmed that lipid scrambling through the scramblase TMEM16F is essential for chemically induced membrane blebbing across cell types, with the kinetics of PS exposure being tightly coupled to the kinetics of bleb formation. Measurement of lipid packing with environment-sensitive probes revealed that lipid scrambling changes the physical properties of the PM, reducing lipid packing and facilitating the bilayer bending required for bleb formation. Accordingly, reducing lipid packing of the PM through cholesterol extraction, elevated temperature, or treatment with biological amphiphiles promoted blebbing in the absence of TMEM16F. Consistent with these cellular observations, blebbing in Caenorhabditis elegans embryos measured via EV production was significantly reduced by depleting the TMEM16-homolog ANOH-2. Our findings suggest that changing membrane biophysical properties by lipid scrambling is an important contributor to the formation of blebs and EVs and potentially other cellular processes involving PM deformation.
Vinculin is an essential structural adaptor protein that localizes to sites of adhesion and is involved in a number of cell processes including adhesion, spreading, motility, force transduction, and cell survival. The...
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Vinculin is an essential structural adaptor protein that localizes to sites of adhesion and is involved in a number of cell processes including adhesion, spreading, motility, force transduction, and cell survival. The C-terminal vinculin tail domain (Vt) contains the necessary structural components to bind and cross-link actin filaments. Actin binding to Vt induces a conformational change that promotes dimerization through the C-terminal hairpin of Vt and enables actin filament cross-linking. Here we show that Src phosphorylation of Y1065 within the C-terminal hairpin regulates Vt-mediated actin bundling and provide a detailed characterization of Y1065 mutations. Furthermore, we show that phosphorylation at Y1065 plays a role in cell spreading and the response to the application of mechanical force.
In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assi...
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In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal;few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29 · A41 base-pair in the anticodon stem with C29 · G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3′ end of the anticodon (position 36) reduced the aminoacylation efficiency ( Vmax Km) of tRNAVal by three orders of magnitude. Base replacement at the 5′ end of
Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis...
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