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It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca2+ signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca2+, ER Ca2+ release is the source of intracellular Ca2+ oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca2+ release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca2+ permeable channels on the plasma membrane, specifically TRPM7. However, Ca2+ influx at the plasma membrane via mechanosensitive Ca2+ permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane., BN/Bionanoscience, Applied Sciences

RecA proteins form a long stable filament on a single-stranded DNA and catalyze strand exchange reaction. The stability of RecA filament changes dramatically with pH, yet its detailed mechanism is not known. Here, using a single molecule assay, we determined the binding and dissociation rates of RecA monomers at the filament ends at various pH. The pH-induced rate changes were moderate but occurred in opposite directions for binding and dissociation, resulting in a substantial increase in filament stability in lower pH. The highly charged residues in C-terminal domain do not contribute to the pH dependent stability. The stability enhancement of RecA filament in low pH may help the cell to cope with acidic stress by fine-tuning of the binding and dissociation rates without losing the highly dynamic nature of the filament required for strand exchange., BN/Bionanoscience, Applied Sciences

It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca2+ signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca2+, ER Ca2+ release is the source of intracellular Ca2+ oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca2+ release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca2+ permeable channels on the plasma membrane, specifically TRPM7. However, Ca2+ influx at the plasma membrane via mechanosensitive Ca2+ permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane., BN/Bionanoscience, Applied Sciences

RecA proteins form a long stable filament on a single-stranded DNA and catalyze strand exchange reaction. The stability of RecA filament changes dramatically with pH, yet its detailed mechanism is not known. Here, using a single molecule assay, we determined the binding and dissociation rates of RecA monomers at the filament ends at various pH. The pH-induced rate changes were moderate but occurred in opposite directions for binding and dissociation, resulting in a substantial increase in filament stability in lower pH. The highly charged residues in C-terminal domain do not contribute to the pH dependent stability. The stability enhancement of RecA filament in low pH may help the cell to cope with acidic stress by fine-tuning of the binding and dissociation rates without losing the highly dynamic nature of the filament required for strand exchange., BN/Bionanoscience, Applied Sciences

A is a DNA-dependent ATPase and mediates homologous recombination by first forming a filament on a single-stranded (ss) DNA. RecA binds preferentially to TGG repeat sequence, which resembles the recombination hot spot Chi (5'-GCTGGTGG-3') and is the most frequent pattern (GTG) of the codon usage in Escherichia coli. Because of the highly dynamic nature of RecA filament formation, which consists of filament nucleation, growth and shrinkage, we need experimental approaches that can resolve each of these processes separately to gain detailed insights into the molecular mechanism of sequence preference. By using a single-molecule fluorescence assay, we examined the effect of sequence on individual stages of nucleation, monomer binding and dissociation. We found that RecA does not recognize the Chi sequence as a nucleation site. In contrast, we observed that it is the reduced monomer dissociation that mainly determines the high filament stability on TGG repeats. This sequence dependence of monomer dissociation is well-correlated with that of ATP hydrolysis, suggesting that DNA sequence dictates filament stability through modulation of ATP hydrolysis., BN/Bionanoscience, Applied Sciences

A is a DNA-dependent ATPase and mediates homologous recombination by first forming a filament on a single-stranded (ss) DNA. RecA binds preferentially to TGG repeat sequence, which resembles the recombination hot spot Chi (5'-GCTGGTGG-3') and is the most frequent pattern (GTG) of the codon usage in Escherichia coli. Because of the highly dynamic nature of RecA filament formation, which consists of filament nucleation, growth and shrinkage, we need experimental approaches that can resolve each of these processes separately to gain detailed insights into the molecular mechanism of sequence preference. By using a single-molecule fluorescence assay, we examined the effect of sequence on individual stages of nucleation, monomer binding and dissociation. We found that RecA does not recognize the Chi sequence as a nucleation site. In contrast, we observed that it is the reduced monomer dissociation that mainly determines the high filament stability on TGG repeats. This sequence dependence of monomer dissociation is well-correlated with that of ATP hydrolysis, suggesting that DNA sequence dictates filament stability through modulation of ATP hydrolysis., BN/Bionanoscience, Applied Sciences