PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling
- University of Oxford, United Kingdom
- University of Glasgow, United Kingdom
- University of Groningen, The Netherlands
- University of Padova, Italy
- University of Gothenburg, Sweden
Figures
Figure 1 with 2 supplements
PDE2A2 localises to the mitochondria in NRVM and regulates mitochondria morphology via DRP1.
(A) Localisation of PDE2A1-GFP, PDE2A2-GFP and PDE2A3-GFP in neonatal rat ventricular myocytes (NRVM) labelled with mitotracker red. Scale bar: 10 µm. (B) Localisation of wild type (PDE2A2wt-RFP) or …
Figure 1—figure supplement 1
PDE2A2 regulates mitochondria morphology in H9C2 myoblasts.
(A) Analysis of mitochondrial length in H9C2 cells treated with Bay 60–7550 (1 µM) or Forskolin (25 µM) and IBMX (100 µM). (B) Left panel: analysis of mitochondrial length in H9C2 cells expressing …
Figure 1—figure supplement 2
The effect of PDE2A inhibition on DRP1 phosphorylation at ser616.
Western blotting analysis of cell lysates from NRVM treated with DMSO, Bay 60–7550 (1 µM) or forskolin (25 µM) plus IBMX (100 µM) and probed for the CDK1 site ser616 on DRP1. The panel on the right …
Figure 2
Mitochondria morphology is affected by PDE2A knock out.
(A) Wild type (MEFwt) and PDE2KO (MEFPDE2KO) mouse embryonic fibroblasts stained with mitotracker green. Scale bar: 10 µm. (B) MEFwt and MEFPDE2KO expressing catalytically inactive (PDE2A2dn-RFP) or …
Figure 3 with 2 supplements
PDE2A2 controls cAMP at the outer mitochondrial membrane.
(A) FRET analysis in NRVM expressing the cytosolic EPAC-based H90 sensor (left) and treated with the PDE2 selective inhibitor Bay 60–7550 (BAY60, 1 µM) or the PDE3 selective inhibitor cilostamide …
Figure 3—figure supplement 1
Localisation of cytosolic and targeted H90.
Representative Western blotting analysis of cytosolic and mitochondrial (Mito) sub-fractions obtained from NRVM lysates not treated and treated with Proteinase K (PK, 10 µM). Cells were not …
Figure 3—figure supplement 2
Effect of PDE2A inhibition on cAMP levels in the matrix.
Representative kinetics (left) and summary data (right) of FRET analysis of cAMP changes detected in the mitochondrial matrix of NRVM expressing the matrix-targeted sensor matrix-H90. Cells were …
Figure 4 with 1 supplement
Submitochondrial localisation of PDE2A2.
(A) Representative Western blotting analysis of cytosolic and mitochondrial sub-fractions obtained from NRVM lysates not treated and treated with Proteinase K (10 µM). PDE2A was probed with a PDE2A …
Figure 4—figure supplement 1
Relative localisation of PDE2A2 and either TOM20 or cytochrome c as revealed by STED microscopy analysis.
(A) Representative confocal and STED images of a HeLa cell labelled with antibodies specific to cytochrome c and TOM20. (B) Representative confocal and STED images of HeLa cell expressing PDE2A2-GFP …
Figure 5 with 1 supplement
PDE2A inhibition affects mitochondrial membrane potential and cell survival.
(A) Left panel: TMRM fluorescence signal before and after application of 1 µM FCCP to NRVM. Representative kinetics (middle panel) and summary of experiments (right panel, n = 60 cells from three …
Figure 5—figure supplement 1
PDE2A affects mitochondrial membrane potential in H9C2 cells.
Mitochondria membrane potential measured in H9C2 myoblasts treated with DMSO, Bay 60–7550 (1 µM) or forskolin (25 µM) plus IBMX (100 µM). The cells were loaded with TMRM to fluorescently label the …
Figure 6
The effect of PDE2A2 inhibition on mitochondria morphology, mitochondrial membrane potential and apoptotic cell death is independent of sAC.
(A) On the left, representative image of MEFwt and MEFsACKO cells either untreated (DMSO) or treated with Bay 60–7550 (1 µM) or cilostamide (10 µM), and loaded with mitotracker red. The summary …
Author response image 1
