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Figure 1 Subcellularly targeted excitation-ratiometric adenosine monophosphate–activated pro- tein kinase activity reporters <t>(ExRai</t> <t>AMPKAR).(A)</t> ExRai AMPKAR consists of an AMPK substrate peptide (MRRVATLVD, AMPKsub), fused to circularly permutated enhanced green fluorescent pro- tein (cpEGFP), followed by a forkhead-associated (FHA1) phosphoamino acid–binding domain. In the absence of AMPK activity, cpEGFP has a preferential excitation at 400 nm with emission at 520 nm. Upon induction of AMPK activity, AMPK phosphorylates the AMPKsub. Phosphorylation induces binding of FHA1 to AMPKsub. This conformational change shifts the excitation of cpEGFP from 400 to 480 nm, while maintaining emission at 520 nm. The change in AMPK activity can be read out as the ratio of 480 nm excitation-emission over 400 nm excitation-emission. (B) In the top panel, a representative image of diffusive ExRai AMPKAR expressed in mouse embryonic fibrob- lasts (MEFs) is shown.In the middle panel, a representative image of mitochondrial-localized ExRai AMPKAR expressed in MEFs is shown. ExRai AMPKAR was localized to the outer mitochon- drial membrane through N-terminal fusion of a localization sequence derived from dual-specific A kinase–anchoring protein (DAKAP, AIQLRSLFPLALPGMLALLGWWWFFSRKKADP). In the bot- tom panel, a representative image of lysosomal-localized ExRai AMPKAR expressed in MEFs is shown.ExRai AMPKAR was localized to the outer lysosomal membrane through N-terminus fusion to lysosome associated membrane protein 1 (LAMP1). Scale bars = 20 nm. Adapted and reprinted from Schmitt et al. (2022) under Creative Commons Attribution 4.0 International License.
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Figure 1 Subcellularly targeted excitation-ratiometric adenosine monophosphate–activated pro- tein kinase activity reporters (ExRai AMPKAR).(A) ExRai AMPKAR consists of an AMPK substrate peptide (MRRVATLVD, AMPKsub), fused to circularly permutated enhanced green fluorescent pro- tein (cpEGFP), followed by a forkhead-associated (FHA1) phosphoamino acid–binding domain. In the absence of AMPK activity, cpEGFP has a preferential excitation at 400 nm with emission at 520 nm. Upon induction of AMPK activity, AMPK phosphorylates the AMPKsub. Phosphorylation induces binding of FHA1 to AMPKsub. This conformational change shifts the excitation of cpEGFP from 400 to 480 nm, while maintaining emission at 520 nm. The change in AMPK activity can be read out as the ratio of 480 nm excitation-emission over 400 nm excitation-emission. (B) In the top panel, a representative image of diffusive ExRai AMPKAR expressed in mouse embryonic fibrob- lasts (MEFs) is shown.In the middle panel, a representative image of mitochondrial-localized ExRai AMPKAR expressed in MEFs is shown. ExRai AMPKAR was localized to the outer mitochon- drial membrane through N-terminal fusion of a localization sequence derived from dual-specific A kinase–anchoring protein (DAKAP, AIQLRSLFPLALPGMLALLGWWWFFSRKKADP). In the bot- tom panel, a representative image of lysosomal-localized ExRai AMPKAR expressed in MEFs is shown.ExRai AMPKAR was localized to the outer lysosomal membrane through N-terminus fusion to lysosome associated membrane protein 1 (LAMP1). Scale bars = 20 nm. Adapted and reprinted from Schmitt et al. (2022) under Creative Commons Attribution 4.0 International License.

Journal: Current protocols

Article Title: Imaging Subcellular AMPK Activity Using an Excitation-Ratiometric AMPK Activity Reporter.

doi: 10.1002/cpz1.771

Figure Lengend Snippet: Figure 1 Subcellularly targeted excitation-ratiometric adenosine monophosphate–activated pro- tein kinase activity reporters (ExRai AMPKAR).(A) ExRai AMPKAR consists of an AMPK substrate peptide (MRRVATLVD, AMPKsub), fused to circularly permutated enhanced green fluorescent pro- tein (cpEGFP), followed by a forkhead-associated (FHA1) phosphoamino acid–binding domain. In the absence of AMPK activity, cpEGFP has a preferential excitation at 400 nm with emission at 520 nm. Upon induction of AMPK activity, AMPK phosphorylates the AMPKsub. Phosphorylation induces binding of FHA1 to AMPKsub. This conformational change shifts the excitation of cpEGFP from 400 to 480 nm, while maintaining emission at 520 nm. The change in AMPK activity can be read out as the ratio of 480 nm excitation-emission over 400 nm excitation-emission. (B) In the top panel, a representative image of diffusive ExRai AMPKAR expressed in mouse embryonic fibrob- lasts (MEFs) is shown.In the middle panel, a representative image of mitochondrial-localized ExRai AMPKAR expressed in MEFs is shown. ExRai AMPKAR was localized to the outer mitochon- drial membrane through N-terminal fusion of a localization sequence derived from dual-specific A kinase–anchoring protein (DAKAP, AIQLRSLFPLALPGMLALLGWWWFFSRKKADP). In the bot- tom panel, a representative image of lysosomal-localized ExRai AMPKAR expressed in MEFs is shown.ExRai AMPKAR was localized to the outer lysosomal membrane through N-terminus fusion to lysosome associated membrane protein 1 (LAMP1). Scale bars = 20 nm. Adapted and reprinted from Schmitt et al. (2022) under Creative Commons Attribution 4.0 International License.

Article Snippet: Materials MEFs (e.g., ATCC, cat. no. CRL-2991) MEF culture medium (see recipe) Opti-MEM I Reduced Serum Medium (e.g., Gibco, cat. no. 31985070) Plasmid DNA encoding ExRai AMPKAR: Untargeted ExRai AMPKAR (e.g., Addgene, cat. no. 192446) Lysosomal outer membrane–targeted ExRai AMPKAR (lyso-ExRai AMPKAR; e.g., Addgene, cat. no. 192449) Mitochondrial outer membrane–targeted ExRai AMPKAR (mito-ExRai AMPKAR; e.g., Addgene, cat. no. 192448) FuGENE HD (e.g., Promega, cat. no. E2311) Hank’s balanced salt solution (HBSS) imaging buffer (see recipe) Schmitt 5 of 19 Current Protocols 26911299, 2023, 5, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.771, W iley O nline L ibrary on [26/01/2024].

Techniques: Activity Assay, Binding Assay, Phospho-proteomics, Membrane, Sequencing, Derivative Assay

Figure 2 Subcellular adenosine monophosphate–activated protein kinase (AMPK) activity mea- sured by excitation-ratiometric AMPK activity reporter (ExRai AMPKAR). (A) Average response of untargeted ExRai AMPKAR used to measure AMPK activity in the cytoplasm of wildtype (WT, purple) or AMPKα1/2 knockout mouse embryonic fibroblasts (MEFs; AMPKα KO, blue) treated with either 40 mM 2-deoxyglucose (2-DG; left; WT n = 31 cells from four experiments, AMPKα KO n = 23 cells from four experiments) or 500 nM MK-8722 (right; WT n = 18 cells from three experiments, AMPKα KO n = 25 cells from three experiments) along with maximum ra- tio change (R/R; ****p ≤0.0001 using unpaired two-tailed t-test). (B) Average response of mitochondria-targeted ExRai AMPKAR in WT (purple) or AMPKα KO (blue) treated with either 40 mM 2-DG (left; WT n = 10 cells from five experiments, AMPKα KO n = 7 cells from three experiments) or 500 mM MK-8722 (right; WT n = 11 cells from four experiments, AMPKα KO n = 11 cells from three experiments) along with R/R (**p = 0.0012, ****p < 0.0001 using unpaired two-tailed t-test). (C) Average response of lysosome-targeted ExRai AMPKAR in WT (purple) or AMPKα KO MEFs (blue) treated with either 40 mM 2-DG (left; WT n = 5 cells from two experiments, AMPKα KO n = 6 cells from two experiments) or 500 mM MK-8722 (right; WT n = 12 cells from three experiments, AMPKα KO n = 8 cells from three experiments) along with

Journal: Current protocols

Article Title: Imaging Subcellular AMPK Activity Using an Excitation-Ratiometric AMPK Activity Reporter.

doi: 10.1002/cpz1.771

Figure Lengend Snippet: Figure 2 Subcellular adenosine monophosphate–activated protein kinase (AMPK) activity mea- sured by excitation-ratiometric AMPK activity reporter (ExRai AMPKAR). (A) Average response of untargeted ExRai AMPKAR used to measure AMPK activity in the cytoplasm of wildtype (WT, purple) or AMPKα1/2 knockout mouse embryonic fibroblasts (MEFs; AMPKα KO, blue) treated with either 40 mM 2-deoxyglucose (2-DG; left; WT n = 31 cells from four experiments, AMPKα KO n = 23 cells from four experiments) or 500 nM MK-8722 (right; WT n = 18 cells from three experiments, AMPKα KO n = 25 cells from three experiments) along with maximum ra- tio change (R/R; ****p ≤0.0001 using unpaired two-tailed t-test). (B) Average response of mitochondria-targeted ExRai AMPKAR in WT (purple) or AMPKα KO (blue) treated with either 40 mM 2-DG (left; WT n = 10 cells from five experiments, AMPKα KO n = 7 cells from three experiments) or 500 mM MK-8722 (right; WT n = 11 cells from four experiments, AMPKα KO n = 11 cells from three experiments) along with R/R (**p = 0.0012, ****p < 0.0001 using unpaired two-tailed t-test). (C) Average response of lysosome-targeted ExRai AMPKAR in WT (purple) or AMPKα KO MEFs (blue) treated with either 40 mM 2-DG (left; WT n = 5 cells from two experiments, AMPKα KO n = 6 cells from two experiments) or 500 mM MK-8722 (right; WT n = 12 cells from three experiments, AMPKα KO n = 8 cells from three experiments) along with

Article Snippet: Materials MEFs (e.g., ATCC, cat. no. CRL-2991) MEF culture medium (see recipe) Opti-MEM I Reduced Serum Medium (e.g., Gibco, cat. no. 31985070) Plasmid DNA encoding ExRai AMPKAR: Untargeted ExRai AMPKAR (e.g., Addgene, cat. no. 192446) Lysosomal outer membrane–targeted ExRai AMPKAR (lyso-ExRai AMPKAR; e.g., Addgene, cat. no. 192449) Mitochondrial outer membrane–targeted ExRai AMPKAR (mito-ExRai AMPKAR; e.g., Addgene, cat. no. 192448) FuGENE HD (e.g., Promega, cat. no. E2311) Hank’s balanced salt solution (HBSS) imaging buffer (see recipe) Schmitt 5 of 19 Current Protocols 26911299, 2023, 5, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.771, W iley O nline L ibrary on [26/01/2024].

Techniques: Activity Assay, Knock-Out, Two Tailed Test