qmtq2-atp biosensor  (Addgene inc)

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic representation of the structural domain and three-dimensional (3D) structural models of qmTQ2-ATP biosensor. 3D structure models were generated with Alphafold2 . (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-ATP biosensor in the presence (blue) and absence (black) of 10 mM ATP. (E) The dose-response curve of qmTQ2-ATP biosensor to ATP in solution. The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-ATP biosensor in the presence (filled circle) and absence (open circle) of 10 mM ATP. The data represents means ± SD (n = 5). (G) Specificity of qmTQ2-ATP biosensor to ATP and other nucleotides. Δτ represents the lifetime changes with the presence and absence of nucleotides. The data represents means ± SD (n = 5). (H) Sequential pseudo-color images of HeLa cells expressing qmTQ2-ATP biosensor in response to 20 mM 2-DG. Fluorescence lifetime (τ) with pseudo color, scale bar: 10 μm. (I) Box-whisker plot comparing Δτ in HeLa cells between the untreated control group and the group treated with 2-DG for 30 minutes. Double asterisks indicate p<0.05 by Student’s t-test.

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Generated, Fluorescence, Expressing, Whisker Assay, Control

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic representation of the structural domain and 3D structure models of qmTQ2-cAMP biosensor. 3D structure models by Alphafold2. (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-cAMP biosensor in the presence (blue) and absence (black) of 1 mM cAMP. (E) Dose-responsive curves of qmTQ2-cAMP biosensor for cAMP (blue circles) and cGMP (red squares). The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-cAMP biosensor in the presence (closed circles) and absence (open circles) of 1 mM cAMP. The data represents means ± SD (n = 5). (G-H) Representative images (G) and the time course (H) of fluorescence lifetime changes in response to 50 μM forskolin stimulation in qmTQ2-cAMP biosensor expressing COS7 cells (scale bar: 10 μm) (n = 7). (I-J) Representative images (I) and the time course (J) of fluorescence lifetime changes induced by 100 μM isoproterenol application in qmTQ2-cAMP biosensor expressing COS7 cells (n = 7) (scale bar: 10 μm).

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic domain structure of the qmTQ2-citrate biosensor. (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-citrate biosensor in the presence (blue) and absence (black) of 20 mM citrate. (E) The dose-response curve of the qmTQ2-citrate biosensor to citrate in solution. The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-citrate biosensor in the presence and absence of 20 mM citrate. The data represents means ± SD (n = 5). (G) Specificity of qmTQ2-citrate biosensor to citrate and other metabolites. Δτ represents the dynamic range obtained with the presence and absence of 20 mM metabolites in TBS buffer. The data represents means ± SD (n = 5). (H) Schematic domain structure of qmTQ2-glucose biosensor. (I–K) The emission and excitation spectra (I), the absorption spectra (J) and the fluorescence decay curve (K) of qmTQ2-glucose biosensor in the presence (blue) and absence (black) of 10 mM glucose. (L) Dose-responsive curve of qmTQ2-glucose biosensor to glucose in solution. The data represents means ± SD (n = 5). (M) Effect of pH on the fluorescence lifetime of qmTQ2-glucose biosensor in the presence and absence of 10 mM glucose. The data represents means ± SD (n = 5). (N) Specificity of qmTQ2-glucose biosensor to monosaccharides and glucose metabolism-related molecules. Δτ represents the dynamic range obtained with the presence and absence of 150 μM these molecules. The data represents means ± SD (n = 5).

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Sequential pseudo-color images of HeLa cells co-expressing qmTQ2-cAMP and RCaMP1h biosensors in response to simultaneous 50 µM histamine dihydrochloride and 100 µM isoproterenol treatment. (B–C) Typical time course (B) and average trace (C) of fluorescence lifetime changes of qmTQ2-cAMP and RCaMP1h biosensors in response to simultaneous histamine and isoproterenol stimulation (n = 12). (D–E) Inhibition of histamine induced calcium oscillations by isoproterenol. Time course of HeLa cells stimulated with 10 μM histamine solely (C) or after pre-treatment with 100 μM isoproterenol (D). Thick lines represent the average traces with qmTQ2-cAMP biosensor shown in purple and the RCaMP1h biosensor in gray. and colorful thin lines represent individual traces (n = 15), scale bar:10 μm.

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Expressing, Fluorescence, Inhibition

Journal: bioRxiv

Article Title: Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

doi: 10.1101/2023.07.26.550743

Figure Lengend Snippet: (A) A scheme of the p33 IDR1 mutant (p33-IDR1m1). (B) Top images: confocal images of droplets formed by either WT eGFP-IDR1 or eGFP-IDR1m1. Note that we used the same eGFP-IDR1 as shown in . Bottom left panel shows the reduced droplet number whereas bottom right panel shows the distribution of doplet size supported by eGFP-IDR1m1. Red box indicates droplet size of <1.0 μm 2 , green box represents 1.0-4.0 μm 2 , blue box represents the % of droplets of 4.0-8.0 μm 2 . (C) Confocal images show the reduced size of VROs formed in N. benthamiana cells expressing eGFP-p33-IDR1m1 versus WT eGFP-p33. Scale bars represent 10 μm. The sizes of VROs are measured in μm 2 . (D) Reduced ATP production within the VROs formed by p33-IDR1m1 or p33-ΔIDR1 in N. benthamiana cells, which expressed the p33 or mutants fused with an ATP biosensor (ATeam YEMK ). The more intense FRET signals are white and red (between 0.5 to 1.0 ratio), whereas the low FRET signals (0.1 and below) are light blue and dark blue. We show the quantitative FRET values (obtained with ImageJ) for numbers of samples in the graph. (E) Top image: Northern blot analysis demonstrates decreased TBSV replicon (+)RNA accumulation in yeast. The His 6 -tagged WT p33 or p33-IDR1m1, p92 pol and DI-72 replicon RNA were co-expressed from plasmids. The accumulation level of replicon RNA was normalized based on 18S rRNA levels (second panel). Bottom panels: The accumulation of His 6 -p33 or His 6 -p33-IDR1m1 is measured by western blotting and anti-His 6 antibodies. Each experiment was repeated three times.

Article Snippet: ATP biosensor (ATeam YEMK ) was fused to p33 replication protein for targeting to VROs.

Techniques: Mutagenesis, Expressing, Northern Blot, Western Blot

Journal: bioRxiv

Article Title: Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

doi: 10.1101/2023.07.26.550743

Figure Lengend Snippet: We propose that TBSV p33 replication protein induces liquid-liquid phase separation of the co-opted, highly concentrated glycolytic and fermentation enzymes and Rpn11, forming unique gel-like vir-condensates associated with VROs, which consist of clustered peroxisomes. The enlarged panel shows that TBSV p33 replication protein is present both in the membranous part of VRO via the N-terminal two transmembrane domains and in the vir-condensate via the C-terminal (cytosol-exposed) region. We propose that co-opted ER membranes and actin filaments provide physical barriers, which facilitate in keeping the virus-induced vir-condensate (in red) and the co-opted clustered peroxisomes within the VRO boundary. A major function of the vir-condensate containing the co-opted glycolytic and fermentation enzymes is to efficiently produce ATP in situ within the VROs. The p33 replication protein is the main organizer by connecting the co-opted membranous substructure and vir-condensate within VRO. CIRV p36 replication protein has similar functions to the TBSV p33 replication protein, except the VROs consist of clustered mitochondria. Abbreviations: VRC: viral replicase complex; vMCS: virus-induced membrane contact site.

Article Snippet: ATP biosensor (ATeam YEMK ) was fused to p33 replication protein for targeting to VROs.

Techniques: In Situ