Visualization of endogenous G proteins on endosomes and other organelles
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, United States
Figures
Figure 1 with 1 supplement
Validation of mNG-β1 and HiBit-β1 cells.
(A) Cartoon showing the peptide tag complementation systems used to label endogenous Gβ1 subunits. (B) SDS-PAGE of HiBit-β1 and mNG-β1 cell lysates; the predicted molecular weights of the edited gene products are 38.9 and 41.1 kilodaltons (KDa), respectively; representative of 3 independent experiments. (C) In permeabilized nucleotide-depleted cells BRET between dopamine D2R-Nluc receptors and mNG-β1-containing heterotrimers increases in response to dopamine (DA; 100 μM) and reverses after addition of GDP (100 μM); mean ± 95% CI; n=27 replicates from two independent experiments. (D) In intact cells BRET between HiBit-β1 and the Gβγ sensor memGRKct-Venus increases after stimulation of D2R dopamine, β2AR adrenergic, or M3R acetylcholine receptors with DA (100 μM), isoproterenol (Iso; 10 μM) and acetylcholine (Ach; 100 μM), respectively. Signals reversed when receptors were blocked with haloperidol (10 μM), ICI 118551 (10 μM) or atropine (10 μM); mean ± 95% CI; n=16 replicates from four independent experiments.
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Figure 1—source data 1
PDF file containing original HiBit blot shown in panel B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data1-v1.pdf
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Figure 1—source data 2
Original files for HiBit blot shown in panel B.
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data2-v1.zip
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Figure 1—source data 3
Numerical data for traces shown in panels C and D.
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
Receptor-mediated accumulation of cyclic AMP (cAMP) is similar in HiBit-β1, mNG-β1 and parent cell lines.
Activation of endogenous β adrenergic receptors with isoproterenol (Iso; 10 μM) produces similar increases in cAMP as indicated by the Nluc-EPAC-VV cAMP sensor in (A) parental HEK and HiBit-β1 cells and (B) parental mNG2(1–10) and mNG-β1 cells; mean ± 95% CI of 20 replicates from n=5 independent experiments.
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Figure 1—figure supplement 1—source data 1
Numerical data for traces shown in panels A and B.
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 3 supplements
Endogenous G proteins are abundant on the plasma membrane but not large organelles.
(A) A single field of view of mNG-β1 cells at three magnifications; scale bars are 40 μm, 20 μm, and 10 μm. (B) mNG-β1 does not colocalize with expressed markers of the endoplasmic reticulum (ER; PTP1b), mitochondria (MT; MOA) or medial-trans Golgi apparatus (GA; GalT); intensity line profiles depict absolute fluorescence intensity in each channel; scale bars are 2 μm.
Figure 2—figure supplement 1
G proteins are not abundant on the endoplasmic reticulum (ER).
Exemplary images of mNG-β1 cells coexpressing the ER marker mRuby2-PTP1b. Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 2—figure supplement 2
G proteins are not abundant on mitochondria (MT).
Exemplary images of mNG-β1 cells coexpressing the MT marker mRuby2-MOA. Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
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Figure 2—figure supplement 2—source data 1
Numerical data for individual line scans (panel C).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
G proteins are not abundant on the medial-trans Golgi apparatus (GA).
Exemplary images of mNG-β1 cells coexpressing the GA marker mRuby2-Golgi-7 (GalT). Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 3 with 7 supplements
Endogenous G proteins colocalize with markers of endosomes and lysosomes.
(A) mNG-β1 colocalizes with expressed markers of early endosomes (EE; FYVE and rab5a), recycling endosomes (RE; rab11a), late endosomes (LE; rab7a) and lysosomes (lyso; LysoView 633); intensity line profiles depict absolute fluorescence intensity in each channel; scale bars are 5 μm. (B) Mean mNG-β1 fluorescence intensity line profiles drawn across the plasma membrane (PM) and FYVE-positive vesicles; mean ±95% CI; n=40 vesicles/cells. (C) mNG-β1 signal/background ratios for regions of interest surrounding the plasma membrane (PM; n=99), FYVE-positive (n=125) and rab5a-positive (n=56) early endosomes, and rab7a-positive (n=26) late endosomes; horizontal lines represent the median. (D) Bystander net BRET signals between HiBit-β1 and Venus-tagged markers of the plasma membrane (PM), endoplasmic reticulum (ER), mitochondria (MT), early endosomes (FYVE and rab5a), recycling endosomes (rab11a) and late endosomes (rab7a); horizontal lines represent the median; n=5–7 independent experiments.
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Figure 3—source data 1
Numerical data for line profiles (panel B), signal/background ratios (panel C) and bystander BRET (panel D).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
G proteins colocalize with the early endosome marker FYVE on some endosomes.
Exemplary images of mNG-β1 cells coexpressing the early endosome marker pmCherry-2xFYVE (FYVE). Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 3—figure supplement 2
G proteins colocalize with the early endosome marker rab5a on some endosomes.
Exemplary images of mNG-β1 cells coexpressing the early endosome marker mRuby2-rab5a (rab5a). Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 3—figure supplement 3
G proteins colocalize with the recycling endosome marker rab11a in a perinuclear region.
Exemplary images of mNG-β1 cells coexpressing the recycling endosome marker mCherry-rab11a (rab11a). Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 10 μm.
Figure 3—figure supplement 4
G proteins colocalize with the late endosome marker rab7a on many endosomes.
Exemplary images of mNG-β1 cells coexpressing the late endosome marker mCherry-rab7a (rab7a). Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 3—figure supplement 5
G proteins are abundant on lysosomes.
(A) Exemplary images of mNG-β1 cells stained with LysoView 633 (LV633); scale bars are 5 μm. (B) Exemplary images of mNG-β1 cells incubated overnight with 10,000 m.w. CF 640 dextran (640 dex); scale bars are 5 μm.
Figure 3—figure supplement 6
G proteins colocalize with the lysosome marker LAMP1.
Exemplary images of mNG-β1 cells coexpressing the lysosome marker LAMP1-mScarlet; scale bars are 5 μm.
Figure 3—figure supplement 7
G proteins colocalize with the trans-Golgi marker TGNP.
Exemplary images of mNG-β1 cells coexpressing the trans-Golgi marker mCherry-TGNP. Intensity line profiles depict fluorescence intensity normalized to the maximum value in each channel; scale bars are 5 μm.
Figure 4 with 2 supplements
Constitutive G protein endocytosis is inefficient.
(A) mNG-β1 colocalizes with newly internalized endocytic vesicles labeled with FM4-64 and CellMask Deep Red (arrowheads); scale bar is 2 μm. (B) A fluorescence intensity line profile for mNG-β1, FM4-64 and CellMask normalized to the peak value of each label at the plasma membrane (PM). (C) Mean mNG-β1, FM4-64 and CellMask fluorescence intensity line profiles drawn across vesicles, normalized to fluorescence intensity at the plasma membrane for each label; mean ± 95% CI; n=45 vesicles/cells.
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Figure 4—source data 1
Numerical data for line profiles (panel C).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Constitutive endocytosis of G proteins is inefficient.
(A) Images of mNG-β1 cells immediately after and 10 minutes after staining with FM4-64. (B) An absolute fluorescence intensity line profile for mNG-β1 and FM4-64; scale bar is 2 μm. In this example absolute fluorescence intensity at the plasma membrane (PM) was similar for the two labels.
Figure 4—figure supplement 2
mNG-HRas ct is less abundant on endocytic vesicles than the plasma membrane.
(A) mNG-HRas ct colocalizes with newly internalized endocytic vesicles labeled with FM4-64 (arrowheads); scale bar is 2 μm. (B) A fluorescence intensity line profile for mNG-HRas ct and FM4-64 normalized to the peak value of each label at the plasma membrane (PM). (C) Mean mNG-HRas ct and FM4-64 fluorescence intensity line profiles drawn across vesicles, normalized to fluorescence intensity at the plasma membrane for each label; mean ±95% CI; n=78 vesicles/cells.
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Figure 4—figure supplement 2—source data 1
Numerical data for line profiles (panel C).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig4-figsupp2-data1-v1.xlsx
Figure 5 with 2 supplements
Receptor activation does not change G protein endocytosis.
(A) mNG-β1 colocalizes with newly internalized endocytic vesicles labeled with FM4-64 and SNAP-tagged β2 adrenergic receptor (β2AR) labeled with Alexa Fluor 674; scale bar is 5 μm. Cells were stimulated with 10 μM isoproterenol for 15 min to induce β2AR internalization. (B) A fluorescence intensity line profile for mNG-β1, FM4-64 and β2AR normalized to the peak value of each label at the plasma membrane (PM). (C) Mean mNG-β1, FM4-64 and β2AR fluorescence intensity line profiles drawn across vesicles, normalized to fluorescence intensity at the plasma membrane for each marker; mean ± 95% CI; n=91 vesicles/cells. (D) Normalized peak mNG-β1/FM4-64 did not differ between vesicles that contained receptors (R; n=91) and vesicles formed by constitutive endocytosis (no R; n=45); n.s., not significant, p=0.20, unpaired t-test. (E) Bystander BRET between HiBit-β1 and markers of early endosomes (EE), recycling endosomes (RE) and late endosomes (LE) was unchanged after 30 minutes of receptor activation with isoproterenol (Iso; 10 μM), dopamine (DA; 100 μM) or acetylcholine (Ach; 100 μM) compared to the DPBS vehicle alone (control); mean ± SD, n=4 independent experiments; no agonist-treated group was significantly different from the control, paired t-test with a false discovery rate (FDR) of 1%.
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Figure 5—source data 1
Numerical data for line profiles (panel C), intensity ratios (panel D) and bystander BRET (panel E).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Transient translocation of endogenous HiBit-β1 from the plasma membrane to intracellular compartments during activation.
(A) Bystander BRET between HiBit-β1 and a Venus-tagged marker of the plasma membrane (PM) decreased, whereas BRET between HiBit-β1 and a Venus-tagged markers of the endoplasmic reticulum (ER), early endosomes (EE), recycling endosomes (RE) and late endosomes (LE) increased during receptor activation with isoproterenol (Iso; 10 μM), dopamine (DA; 100 μM) or acetylcholine (Ach; 100 μM) compared to the DPBS vehicle alone (control); mean ± SD, n=5 independent experiments; all agonist-treated groups were significantly different from the control, paired t-test with a false discovery rate (FDR) of 1%. (B) Time course of bystander BRET between HiBit-β1 and a Venus-tagged marker of the ER during activation of D2R with DA (100 μM), followed by block of D2R with the antagonist haloperidol (Hal; 10 μM); mean ± 95% CI of 24 replicates from n=4 independent experiments.
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Figure 5—figure supplement 1—source data 1
Numerical data for bystander BRET values (panel A) and traces (panel B).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
G protein abundance on endosomes after GPCR and G protein activation.
Bystander BRET between HiBit-β1 and markers of early endosomes (EE), recycling endosomes (RE) and late endosomes (LE) was largely unchanged after 5 min (A) or 15 min (B) of receptor activation with isoproterenol (Iso; 10 μM), dopamine (DA; 100 μM), or acetylcholine (Ach; 100 μM) compared to the DPBS vehicle alone (control); mean ± SD, n=4 independent experiments; only two agonist-treated groups (indicated with *) were significantly different from the control, paired t-test with a false discovery rate (FDR) of 1%.
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Figure 5—figure supplement 2—source data 1
Numerical data for bystander BRET (panels A and B).
- https://cdn.elifesciences.org/articles/97033/elife-97033-fig5-figsupp2-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | GNB1 | GenBank | Gene ID: 2782 | Gene (Homo sapiens) |
| Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573; RRID:CVCL_0045 | Cell line (Homo sapiens) |
| Cell line (Homo sapiens) | HEK293T expressing mNG2(1–10) | PMID:35271311 | Obtained from Manuel Leonetti | |
| Cell line (Homo sapiens) | HiBit-β1 | This paper | See Materials and Methods | |
| Cell line (Homo sapiens) | mNG-β1 | This paper | See Materials and Methods | |
| Sequence-based reagent | crRNA | Integrated DNA Technologies | See Materials and Methods | |
| Sequence-based reagent | tracrRNA | Integrated DNA Technologies | See Materials and Methods | |
| Sequence-based reagent | ssODN HDR donor | Integrated DNA Technologies | See Materials and Methods | |
| Recombinant protein | Cas9 Nuclease V3 | Integrated DNA Technologies | 1081059 | |
| Recombinant DNA reagent | mRuby-Golgi-7 | Addgene | 55865 | |
| Recombinant DNA reagent | mRuby2-Rab5a-7 | Addgene | 55911 | |
| Recombinant DNA reagent | mCherry-Rab7a-7 | Addgene | 55127 | |
| Recombinant DNA reagent | mCherry-Rab11a-7 | Addgene | 55124 | |
| Recombinant DNA reagent | pmCherry-2xFYVE | Addgene | 140050 | |
| Recombinant DNA reagent | mCherry-TGNP-N-10 | Addgene | 55145 | |
| Recombinant DNA reagent | Lamp1-mScarlet-I | Addgene | 98827 | |
| Recombinant DNA reagent | Venus-2xFYVE | This paper | Venus version of pmCherry-2xFYVE | |
| Recombinant DNA reagent | mRuby2-MOA | This paper | mRuby2 version of Venus-MOA | |
| Recombinant DNA reagent | mRuby2-PTP1b | This paper | mRuby2 version of Venus-PTP1b | |
| Recombinant DNA reagent | Venus-kras | PMID:21364942 | ||
| Recombinant DNA reagent | Venus-PTP1b | PMID:22816793 | ||
| Recombinant DNA reagent | Venus-MOA | PMID:22816793 | ||
| Recombinant DNA reagent | Venus-rab5a | PMID:21364942 | ||
| Recombinant DNA reagent | Venus-rab7a | PMID:27528603 | ||
| Recombinant DNA reagent | Venus-rab11a | PMID:27528603 | ||
| Recombinant DNA reagent | memGRKct-Venus | PMID:19258039 | ||
| Chemical compound | PEI Max | Polysciences | 24765 | |
| Other | CellMask Deep Red | ThermoFisher | C10046 | 1:1,000 |
| Other | LysoView 633 | Biotium | 70058 | 1:1,000 |
| Other | CF 640 dextran | Biotium | 80115 | 25 μg ml–1 |
| Other | FM4-64; SynaptoRed | Sigma-Aldrich | 574799 | 5 μM |
| Software | CRISPResso2 | PMID:30809026 | RRID:SCR_024503 | |
| Software | ImageJ | imagej.net/ij/ | RRID:SCR_003070 | |
| Software | GraphPad Prism | graphpad.com | RRID:SCR_002798 |
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Visualization of endogenous G proteins on endosomes and other organelles
eLife 13:RP97033.
https://doi.org/10.7554/eLife.97033.3
