Journal: Frontiers in Cell and Developmental Biology

Article Title: Peroxisomal ATP Uptake Is Provided by Two Adenine Nucleotide Transporters and the ABCD Transporters

doi: 10.3389/fcell.2021.788921

Figure Lengend Snippet: Comparison of ATP levels determined with FRET-based ATeam reporter proteins in wild-type and mutant yeast strains. Cells were transformed with the different ATeam reporter proteins, grown overnight in oleate medium and transferred to 25 mM MES buffer (pH 6.0) supplemented with 20 g/L glucose prior to measurements. (A) Peroxisomal ATP levels determined with reporter proteins ATeam-per and ATeam-per (mut) (B) ATP levels determined with reporter proteins ATeam-cyt and ATeam-cyt (mut). FRET was determined by measuring 527/475 nm. Ratios were calculated after subtraction of the background signal from wild-type cells transformed with an empty vector. The semi-relative ATP levels were then calculated by subtracting the 572/475 ratio of the ATeam(mut) reporter from the 572/475 of ATeam reporter. Data are means ± SD of values from 4–13 independent experiments. Only most relevant statistic relations are indicated in , all are given in . ****, ***, **, and * indicate significance with a p -value of p < 0.0001, p < 0.001, p < 0.01, and p < 0.05 respectively.

Article Snippet: To provide in vivo evidence for the role of Ant1p and Pxa1p/Pxa2p in peroxisomal ATP uptake, we expressed modified versions of the FRET-based ATeam reporter protein ( ; ) in the different yeast strains to measure the relative ATP levels in the peroxisomes (ATeam-per), i.e. extended with a carboxy-terminal peroxisomal targeting signal) and the cytosol (ATeam-cyt).

Techniques: Comparison, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: PLoS Biology

Article Title: Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

doi: 10.1371/journal.pbio.1002511

Figure Lengend Snippet: (A) Primary cortical neurons were transfected with control or Wfs1 siRNA using the N-TER nanoparticle siRNA transfection system and stained with JC-10, which emits light from 525 nm to 590 nm, depending on mitochondrial membrane potential. Values shown are corrected by subtracting the values obtained in the presence of FCCP incubation (5 μM). The red to green fluorescence ratio demonstrated a slight but significant decrease in the Wfs1 siRNA group. (B) Neurons were transfected with plasmids expressing scrambled shRNA or Wfs1 shRNA, firefly luciferase construct containing NRF2 binding site, and Renilla luciferase. Firefly luciferase signal normalized to Renilla signal demonstrates no change in NRF2 activity. NRF2 overexpression-induced reporter activity was used as a positive control. (C) Neurons transfected with the ATP sensor ATeam and treated with 2-deoxyglucose (12 mM)/oligomycin (2.5 μM) or glutamate (2 mM) (both used as positive controls) show a decrease in relative cytosolic ATP levels. (D) Neurons were transfected with the ATP sensor ATeam and scrambled or Wfs1 shRNAs. WFS1-deficient neurons show a lower cytosolic ATP level as compared to control. ** p < 0.01 and *** p < 0.001 compared with respective control group. Underlying data is shown in .

Article Snippet: Next, we estimated cell ATP levels using the fluorescence resonance energy transfer (FRET)-based ATP sensor ATeam, which employs the epsilon subunit of a bacterial F 0 F 1 -ATPase.

Techniques: Transfection, Control, Staining, Membrane, Incubation, Fluorescence, Expressing, shRNA, Luciferase, Construct, Binding Assay, Activity Assay, Over Expression, Positive Control

Journal: Chemical reviews

Article Title: Genetically-encodable Fluorescent Biosensors for Tracking Signaling Dynamics in Living Cells

doi: 10.1021/cr100002u

Figure Lengend Snippet: Fluorescent biosensors for studying signaling dynamics in living cells.

Article Snippet: Ateam-3.10 , absolute [ATP] i , 313 , FRET-based; K D (ATP): 7.4 μM.

Techniques: Activity Assay, Sequencing, Activation Assay, Pulse Chase, Ligand Binding Assay, Binding Assay, Modification, Residue, Mutagenesis, Membrane, In Vitro, Phospho-proteomics, Methylation, Fluorescence, Ubiquitin Proteomics, Derivative Assay, Imaging