Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.
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This work was supported by a Medical Research Council grant (G0900069) to R.A.N. and C.R.L.T. and a Wellcome Trust Investigator Award (WT095643) to C.R.L.T.
The authors declare no competing financial interests.
Integrated supplementary information
A. Schematic to show the generation of a P2XA deletion construct for use in homologous recombination. 1.6kb downstream and 1.4kb upstream of the P2XA coding sequence were amplified and cloned either side of the floxed blasticidin cassette in pLPBLP. B. Bright field images of cells in KK2, and after 10min and 60min after changing the media from KK2 to water to induce osmotic shock. Wild-type cells round up within 10 minutes, but fully recover to their normal shape within 60 minutes in water. P2XA− cells also round up within 10 minutes, but fail to recover their shape even after 60 minutes in water. P2XA− cells expressing P2XA:GFP behave like wild-type cells. Wild-type cells treated with 10uM copper fail to recover their shape after 60 minutes in water. Scale bar = 20 μm C. Time course of cell rounding and recovery. Wild-type cells (black squares, solid line) round up for 20-30 min and then regain their normal shape by 40 minutes. P2XA− cells (red square, solid line) become rounder than wild type cells and stay round throughout the entire time course. P2XA− cells expressing P2XA:GFP (red square, dashed line) are similar to wild-type cells, but get slightly rounder and take longer to regain their shape. Wild-type cells treated with 10 μM copper (black square, dashed line) stay round throughout the entire time course. Error bars represent s.e.m. and results are means of n = 3 independent experiments, each with 120 cells. Statistical source data for Supplementary Fig 1C can be found in Supplementary Table 2.
Fluorescent images of RFP tagged mutants expressed in wild-type Dictyostelium. Fluorescence shows that P2XA(R63A), P2XA(R285K) and P2XA(K67A/K289A) all correctly localise to the contractile vacuole in Dictyostelium. Scale bar = 5 μm.
Supplementary Figure 3 P2XA is specifically expressed on CV’s at all stages of the cycle and undergoes a ‘ring to patch’ transition
A. Localization of Rab7:GFP and P2XA:RFP in wild-type cells. No co-localization is observed. Scale bar = 5 μm B. Localization of Dajumin:GFP and P2XA:RFP in wild-type cells at the maturation (top panel) and ‘ring to patch’ (bottom panel) stages of the CV cycle. P2XA:RFP co-localizes with Dajumin:GFP during maturation, but localization of P2XA:RFP is restricted to the plasma membrane contact site of the CV during the ring to patch transition stage. Scale bar = 5 μm C. Localization of drainin:GFP and P2XA:RFP in wild-type cells at the maturation (top panel) and ‘ring to patch’ (bottom panel) stages of the CV cycle. P2XA:RFP co-localizes with drainin:GFP at all stages of the cycle. Scale bar = 5 μm D. Localization of rab8a:GFP and P2XA:RFP in wild-type cells. P2XA:RFP co-localizes with rab8a:GFP during the ‘ring to patch’ transition stage of the CV cycle. Scale bar = 5 μm.
A. Confocal images of P2XA-GCaMP2 expressed in HEK cells before (control), during (+ATP) and after (wash) stimulation with ATP (1 mM-10 μM). Increases in fluorescence can be seen when stimulated with 100 μM, 300 μM and 1mM ATP. B. GCaMP2 acts as a sensor for P2XA activity. Fluorescent confocal images of HEK cells expressing P2XA-GCaMP2, P2XA-GFP and P2XA(K67A/K289A)-GCaMP2 over time. Following stimulation with 1mM ATP, P2XA-GCaMP2 cells show a transient increase in fluorescence at 20 seconds, whilst there is no change in fluorescence for P2XA-GFP or P2XA(K67A/K289A)-GCaMP2. Scale bar = 10 μm.
A. Time course of cell rounding and recovery of P2XA− cells expressing P2XA-GCaMP2. Over-expression of P2X-GCaMP2 in the P2XA− background (green) rescues the osmoregulation phenotype so that cells transiently round up after 10 mins in water but are able to recover their shape within 60 mins. Error bars represent s.e.m. and results are means of n = 4 independent experiments. Statistical source data for Supplementary Fig 5A can be found in Supplementary Table 2. B. Fluorescence LUT images of Dictyostelium cells expressing Actin15-GCaMP2 or Actin15-GFP. Increases in fluorescence are observed in cells expressing Actin15-GCAMP2 after ATP stimulation (30 μM) in the presence of extracellular Ca 2+ only. No changes in fluorescence are observed in cells expressing Actin15-GFP in the presence or absence of Ca 2+. Scale bar = 15 μm C. Plot of changes in fluorescence of cells expressing Actin15-GCaMP2 or Actin15-GFP over time following 30 μM ATP stimulation. Error bars represent s.e.m. and results are means of n = 5 independent experiments, each with 5 cells. Increases in fluorescence are observed in cells expressing Actin15-GCaMP2 only in the presence of extracellular Ca 2+.
A. The anti rab11-GTP antibody is specific for rab11-GTP in vitro. Rab11a-GST or Rab8-GST were loaded with either GTPγS or GDP, incubated with anti rab11-GTP antibody and then immunoprecipitated using protein A/G agarose beads. Bound protein was probed with an anti-GST antibody. The anti rab11-GTP antibody could only immunoprecipitate bacterially expressed rab11a when loaded with GTP. B. The anti rab11-GTP antibody is specific for rab11a-GTP in vivo. Immunoprecipitation of rab11a-GTP from wild-type cells expressing Rab11a-RFP, Rab7-RFP or Rab8-RFP. Lysate from each strain was incubated with anti rab11-GTP antibody and immunoprecipitated using protein A/G agarose beads. Bound and unbound proteins were probed with an anti RFP antibody. (I = input, B = bound, NB = not bound). The anti rab11-GTP antibody could only immunoprecipitate RFP from cells expressing rab11a-RFP. C. The anti rab11-GTP antibody works in the linear range for protocols used throughout the manuscript. Top panel: Lysate from AX4 cells expressing rab11a-RFP was used neat, at a 1:2 dilution or a 1:4 dilution for immunoprecipitation of rab11a-GTP using the anti rab11-GTP antibody. Bottom panel: Densitometry analyses of bound (rab11a-GTP) and unbound (rab11a-GDP) bands reveal that the GTP/GDP ratios remain constant for each sample dilution. Error bars represent s.e.m. and results are means of n = 3 independent experiments. Statistical source data for Supplementary Fig 6C can be found in Supplementary Table 2.
Supplementary Figure 7 An evolutionarily conserved group of Rab GAPs containing a TBC Rab GAP domain and EF hands.
A conserved group of Rab GAPs containing a TBC Rab GAP domain and EF hands from different phyla. Proteins from D. discoidium (CnrF, DDB_G0295717 and DDB_G0275421), S. pombe (NP_596678), P. tetraurelia (XP_001441542), D. rerio (NP_001120987), X. tropicalis (NP_001107313), M. musculus (NP_001104774), H. sapiens (NP_942568) are shown. A. Protein structure is conserved throughout evolution. B. Alignment of the TBC Rab GAP domain. Homologous sequences were identified using the conserved domains database (Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, Deweese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 2011 Jan;39(Database issue):D225-9. Epub 2010 Nov 24. [PubMed PMID: 21109532]). The alignment was computed using MUSCLE requesting 16 iterations (MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004, 32:1792–1797.). The aligned TBC Rab GAP domain was extracted using CLC Genomics workbench (www.CLCbio.com). C. Alignment of the EF hand domain. Homologous sequences were identified using the conserved domains database (CDD ref). The alignment was computed using MUSCLE requesting 16 iterations (MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004, 32:1792–1797.). The aligned EF hand domain was extracted using CLC Genomics workbench (www.CLCbio.com).
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Live cell fluorescence imaging of a wild-type cell expressing dajumin:GFP during CV cycling (imaged between 0 and 330 seconds after osmotic shock). (MOV 521 kb)
Live cell fluorescence imaging of a P2XA− cell expressing dajumin:GFP during CV cycling (imaged between 60 and 210 seconds after osmotic shock). (AVI 28780 kb)
Live cell fluorescence imaging of a wild-type cell expressing drainin:GFP during CV cycling (imaged between 60 and 190 seconds after osmotic shock). (MOV 468 kb)
Live cell fluorescence imaging of a P2XA− cell expressing drainin:GFP during CV cycling (imaged between 60 and 190 seconds after osmotic shock). (MOV 426 kb)
Live cell fluorescence imaging of a wild-type cell expressing rab8:GFP during CV cycling (imaged between 240 and 350 seconds after osmotic shock). (MOV 546 kb)
Live cell fluorescence imaging of a P2XA− cell expressing rab8:GFP during CV cycling (imaged between 240 and 350 seconds after osmotic shock). (MOV 604 kb)
Live cell fluorescence imaging of a HEK cells expressing P2XA-GCaMP2 and stimulated with ATP (1mM) which causes a transient increase in fluorescence. (AVI 547 kb)
Live cell fluorescence imaging of a HEK cells expressing P2XA-GFP and stimulated with ATP (1mM). No increase in fluorescence is observed. (AVI 410 kb)
Live cell fluorescence imaging of a wild-type cells co-expressing P2XA-GCaMP2 and P2XA-RFP and imaged in water during osmotic shock. (AVI 27057 kb)
Live cell fluorescence imaging of a wild-type cells co-expressing P2XA-GFP and P2XA-RFP and imaged in water during osmotic shock. (AVI 1126 kb)
Live cell fluorescence imaging of a wild-type cells co-expressing mutant (K67A/K289A) P2XA-GCaMP2 and mutant (K67A/K289A) P2XA-RFP and imaged in water during osmotic shock. (AVI 16510 kb)
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Parkinson, K., Baines, A., Keller, T. et al. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel. Nat Cell Biol 16, 87–98 (2014). https://doi.org/10.1038/ncb2887
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