Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast number of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent. Recent single-molecule studies from multiple research groups have uncovered that protein concentration can facilitate the unbinding of DNA-bound proteins, revealing regulation of protein unbinding as another mechanistic paradigm for gene regulation. In this Account, we review these recent in vitro and in vivo single-molecule experiments that uncovered the concentration-facilitated protein unbinding by multiple types of DNA-binding proteins, including sequence-nonspecific DNA-binding proteins (e.g., nucleoid-associated proteins, NAP), sequence-specific DNA-binding proteins (e.g., metal-responsive transcription regulators CueR and ZntR), sequence-neutral single-stranded DNA-binding proteins (e.g., Replication protein A, RPA), and DNA polymerases. For the in vitro experiments, Marko's group investigated the exchange of GFP-tagged DNA-bound NAPs with nontagged NAPs in solution of increasing concentration using single-molecule magnetic-tweezers fluorescence microscopy. The faster fluorescence intensity decrease with higher nontagged NAP concentrations suggests that DNA-bound NAPs undergo faster exchange with higher free NAP concentrations. Chen's group used single-molecule fluorescence resonance energy transfer measurements to study the unbinding of CueR from its cognate oligomeric DNA. The average microscopic dwell times of DNA-bound states become shorter with increasing CueR concentrations in the surroundings, demonstrating that free CueR proteins can facilitate the unbinding of the incumbent one on DNA through either assisted dissociation or direct substitution. Greene's group studied the unbinding of RPAs from single-stranded DNA using total internal reflection fluorescence microscopy and DNA curtain techniques. The fluorescence intensity versus time traces show faster decay with higher wild-type RPA concentrations, indicating that DNA-bound RPAs can undergo a concentration-facilitated exchange when encountering excess free RPA. van Oijen's group investigated the leading/lagging-strand polymerase exchange events in the bacteriophage T7 and E. coli replication systems using a combination of single-molecule fluorescence microscopy and DNA-flow-stretching assay. The processivity was observed to have larger decrease when the concentration of the Y526F polymerase mutant increases, indicating that the unbinding of the polymerase is also concentration-dependent. Using stroboscopic imaging and single-molecule tracking, Chen's group further advanced their study into living bacterial ...

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Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions—however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that protects the cells from radical oxygen species. To study the behavior of endogenous SOD1 under a microscope, we genetically modified H1 human embryonic stem cells (hESCs) to express SOD1 fused with a SNAP-tag, a protein tag that can be covalently labeled with a variety of synthetic probes. The engineered homozygous clone expressing SOD1-SNAP fusion proteins has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro , providing a versatile platform for imaging-based studies of SOD1.

Despite its abuse potential, nicotine, acting on nicotinic acetylcholine receptor, has possible medicinal uses, in particular in treating neuro degenerative diseases. Therefore, animal models to evaluate exposure need to be characterized. Administration via drinking water is a stressfree route of administration but often results in low blood nicotine levels. Here we evaluated chronic exposure to low, medium and high concentrations of nicotine in drinking water. Three-month-old C57BL/6 male mice were treated for 23 days with 20, 120 or 300 µg/ml nicotine in 2% saccharin water, corresponding to 5, 30 and 55 mg/kg/d, respectively. Food intake and body weight were monitored, blood nicotine and cotinine levels, and 125I-epibatidine-binding sites were determined at day 23.Average blood cotinine levels of 11.7, 151.8 and 192.0 ng/ ml were detected in mice receiving the low, medium and high dose, respectively. In contrast, nicotine was only consistently measured in the group receiving 300 µg/ml, with an average blood level of 6.2 ng/ml and was the only treatment group to exhibit significantly decreased food intake (p=0.005) and body weight (p=0.043), as well as increased I125-epibatidine binding in cortex (p=0.055) and hippocampus (p = 0.019). We evaluated possible effects of chronic nicotine (300µg/ml) exposure on anxiety-like behavior using the open field test. An anxiolytic effect was found compared to controls and there was no evidence for anxiogenic effects of chronic nicotine. Thus, a high concentration of nicotine in drinking water was necessary to achieve consistent blood nicotine levels in mice, which correlated with markers considered hallmarks of chronic nicotine treatment.