Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
- University of Copenhagen, Denmark
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Germany
- European Molecular Biology Laboratory, Germany
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- AstraZeneca, United Kingdom
- University of Washington, United States
- Saarland University, Germany
Figures
Figure 1
Synthesis of membrane-permeant and photoactivatable PI(4,5)P2 (cg-PI(4,5)P2).
(a) Synthesis of PI(4,5)P2 derivatives 1a,b and 2a,b. Reagents and conditions: (a) CH2Cl2:HCO2H 4:1, room temperature (rt), 3 hr, 88%; (b) (FmO)2P-NiPr27, 1H-tetrazole, CH2Cl2, rt, 1 hr, then AcO2H, …
Figure 2 with 1 supplement
Characterization of PI(4,5)P2 UV uncaging in-vitro, loading of cg-PI(4,5)P2 into living cells and visualization of PI(4,5)P2 uncaging in several cell types.
(a) Uncaging of cg-PI(4,5)P2 micelles on a glass coverslip results in the relocation of a high affinity PI(4,5)P2 sensor, PLCδ1-PH-EGFP, to micelles following UV light exposure, as seen by a local …
Figure 2—figure supplement 1
PI(4,5)P2 uncaging elevates plasmalemmal PI(4,5)P2.
(a) Confocal imaging of elevated PI(4,5)P2 levels following uncaging of 1a,b. These tsA-201 cells overexpress M1 receptors and the PI(4,5)P2-fluorescence sensor PLCδ1-PH-RFP. Due to the high …
Figure 3
PI(4,5)P2 uncaging increases actin levels near the plasma membrane and recruits the low affinity PI(4,5)P2 sensor PLCδ4-PH-EGFP to plasma membranes of adrenal chromaffin cells.
(a) TIRF imaging of HEK cell footprints transfected with lifeact-RFP to label actin. Cells were either loaded for 30 min at 37°C with 20 µM cg-PI(4,5)P2 (+cg-PI(4,5)P2, top) or not loaded (No …
Figure 4 with 3 supplements
PI(4,5)P2 uncaging potentiates exocytosis in adrenal chromaffin cells, which depends on the lipid head group but does not alter depolarization-induced currents.
(a) Physiological stimulation paradigm to investigate the effect of PI(4,5)P2 uncaging on exocytosis. Cells were loaded with compounds 1a,b or 2a,b prior to experiments. After a pre-pulse of …
Figure 4—figure supplement 1
Incubation with cg-PI(4,5)P2 does not affect exocytosis.
Chromaffin cells were incubated with either 0.02% pluronic (Control) or 0.02% pluronic +25 µM cg-PI(4,5)P2 (+cg-PI(4,5)P2) for 30 min as described earlier. Reliable uptake of cg- PI(4,5)P2 was …
Figure 4—figure supplement 2
Uncaging of PI(4,5)P2 DOG (compound 1a,b in Figure 1a) does not alter depolarization-induced currents.
Depolarization-induced cumulative currents (charges mostly originating from Ca2+-currents) corresponding to the capacitance traces depicted in Figure 4b during the pre-pulse (left) and the test …
Figure 4—figure supplement 3
Uncaging of PI(4,5)P2 does not cause an increase of intracellular [Ca2+] but enhances the rate of single vesicle fusion events.
All cells were loaded with compound 2a,b, (Figure 1) and analyzed following a depolarization pre-pulse protocol as depicted in Figure 4a (not shown). (a) Measurements (once every second) of …
Figure 5 with 1 supplement
Uncaging of PI(4,5)P2, but not DAG augments exocytosis.
(a) Left panel: mean whole-cell capacitance responses during the test pulse of chromaffin cells loaded with cg-PI(4,5)P2 (data from compounds 1a,b and 2a,b pooled, uncaging group: blue, control …
Figure 5—figure supplement 1
Blocking PI(4,5)P2-degradation to DAG augments recovery of the RRP.
These experiments measure exocytosis (capacitance changes) induced by sudden intracellular Ca2+ elevations. (a) Ca2+ uncaging (at arrow) stimulates fast and slow components of exocytosis. The …
Figure 6 with 1 supplement
Exocytosis potentiation by PI(4,5)P2 uncaging requires synaptotagmin-1 and Munc13-2, but not CAPS.
(a–c) All cells were loaded with cg-PI(4,5)P2 prior to experiments and subjected to the stimulation paradigm shown in Figure 4a. Average whole-cell capacitance responses during the test pulse are …
Figure 6—figure supplement 1
Uncaging cg-PI(4,5)P2 in-between the pre-pulse and the test pulse enhances exocytosis during the test-pulse.
Supplementary data figure of electrophysiological experiments depicted in Figures 5 and 6. (a) Loading protocol. Cells were incubated with cg-PI(4,5)P2 for 30 min leading to reliable uptake of …
Figure 7 with 1 supplement
Uncaging PI(4,5)P2 induces rapid exocytosis.
(a) PI(4,5)P2 uncaging rapidly increased membrane capacitance measured during the first uncaging flash (stimulation protocol: see Figure 4a), indicative of fast vesicle fusion. Averaged capacitance …
Figure 7—figure supplement 1
Fast release of vesicles upon first PI(4,5)P2 uncaging event in wild type chromaffin cells.
Our uncaging protocol in chromaffin cells included four sequential uncaging pulses every 15 s. Figure 7 shows only the average capacitance traces during the first uncaging. Here, the membrane …
Figure 8
Uncaging PI(4,5)P2 distinguishes mechanism of lipid-binding.
Two different roles of lipid-binding proteins can be distinguished by lipid uncaging: protein A (e.g. CAPS) binds to PI(4,5)P2 in order to bring it to the vesicle and fusion machinery (localization …
Chemical structure 1
Synthesis of head group 10a,b.
https://doi.org/10.7554/eLife.30203.018
Chemical structure 2
Synthesis of 1a,b.
https://doi.org/10.7554/eLife.30203.019
Chemical structure 3
Synthesis of 2a,b.
https://doi.org/10.7554/eLife.30203.020
Chemical structure 4
Structure determination of 4- and 5-isomers.
https://doi.org/10.7554/eLife.30203.021
Chemical structure 5
Synthesis of 7-diethylamino-4-hydroxymethyl-2-oxo-2H-chromen 20.
https://doi.org/10.7554/eLife.30203.022Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (Mus.musculus) | CD1 | Department of Experimental Medicine, Faculty of Health and Medical Sciences, Unviersity of Copenhagen. | ||
| genetic reagent (M.musculus) | Syt-1 nul allele (gene symbol: syt1) | Geppert et al., 1994 | PMID: 18308932 | |
| genetic reagent (M.musculus) | Munc13-2 null allele (gene symbol: Unc13b) | Varoqueaux et al., 2002 | PMID: 12070347 | |
| genetic reagent (M.musculus) | CAPS1 null allele (gene symbol: Cadps) | Speidel et al., 2005 | PMID: 15820695 | |
| genetic reagent (M.musculus) | CAPS2 null allele (gene symbol: Cadps2) | Jockusch et al., 2007 | PMID: 18022372 | |
| cell line (HEK 293T) | HEK 293T | ATCC | CRL-1573 | Experiments in Figure 2b |
| cell line (HEK 293T) | HEK 293T | A gift from Dr. Theres Schaub and Prof Victor Tarabykin, Institute of Cell Biology and Cell Biology, Charité Berlin | Experiments Figure 3a | |
| cell line (COS-7) | COS-7 | ATCC | CRL-1651 | |
| cell line (tsA201) | tsA201 | Sigma-aldrich | Sigma-aldrich: 96121229 | |
| transfected lentiviral construct (p156rrl-pCMV- PLCδ4PH-EGFP) | PLCδ4-PH-GFP | This paper | Local reference: 131 | plasmid with PLCδ4 received from Thomas F. J. Martin (Department of Biochemistry, University of Wisconsin) |
| transfected construct (pCMV-PLCδ1-PH-EGFP) | PLCδ1-PH-GFP | Michael Krauss (Leibniz- Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany). | ||
| transfected construct (pCMV-PLCδ1-PH-RFP) | PLCδ1-PH-RFP | Ken Mackie (The Gill Center for Biomolecular Science, Bloomington, Indiana) | ||
| transfected construct (pCMV-mcherry-INPP5E) | mCh-INPP5E | Posor et al., 2013 | ||
| transfected construct (pCMV-mRFPruby- N1*Lifeact) | lifeact-RFP | Geerd van den Bogaart (Radboud University Medical Center, Nijmegen, The Netherlands) | PMID: 18536722 | |
| commercial assay or kit | QIAprep Spin Miniprep Kit | Qiagen | ||
| commercial assay or kit | QIAquick Gel Extraction Kit | Qiagen | ||
| commercial assay or kit | QIAquick PCR Purification Kit | Qiagen | ||
| chemical compound, drug | DMSO | Sigma-aldrich | Sigma-aldrich: D8418 | |
| chemical compound, drug | Ascorbic aci | Sigma-aldrich | Sigma-aldrich: A5960 | |
| chemical compound, drug | CaCl2 | Sigma-aldrich | Sigma-aldrich: 499609 | |
| chemical compound, drug | CellMask | Invitrogen | Invitrogen: C10046 | |
| chemical compound, drug | CsOH | Sigma-aldrich | Sigma-aldrich 516988 | |
| chemical compound, drug | DMEM | Gibco/Thermo Fisher | Gibco/Thermo Fisher: 31966047 | Experiments in Figure 3a |
| chemical compound, drug | DMEM | Lonza | Lonza: BE12-741F | Experiments in Figure 2b,c |
| Chemical compound, drug | HBSS | Gibco/Thermo Fisher | 14025–050 | |
| chemical compound, drug | caged DOG-PI(4,5)P2 | This paper | European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany. Att: Carsten Schultz (schultz@embl.de) | |
| chemical compound, drug | EDTA | Sigma-aldrich | Sigma-aldrich: E5134 | |
| chemical compound, drug | Fetal Bovine Serum (FBS) | Gibco/Thermo Fisher | Thermo Fisher/Gibco: 16140063 | Experiments in Figure 3a |
| chemical compound, drug | Fetal Bovine Serum (FBS) | Gibco/Thermo Fisher | Thermo Fisher/Gibco: 10270–106 | Experiments in Figure 2b,c |
| chemical compound, drug | Fura-4F | Invitrogen | Invitrogen: F14174 | |
| chemical compound, drug | Furaptra | Invitrogen | Invitrogen: M1290 | |
| chemical compound, drug | Glucose | Sigma-aldrich | Sigma-aldrich: G8270 | |
| chemical compound, drug | HEPES | Sigma-aldrich | Sigma-aldrich: H3375 | |
| chemical compound, drug | Insulin-transferrin- selenium-X | Invitrogen | Invitrogen: 51500056 | |
| chemical compound, drug | KCl | Sigma-aldrich | Sigma-aldrich: P5405 | |
| chemical compound, drug | L-Cysteine | Sigma-aldrich | Sigma-aldrich: C7352 | |
| chemical compound, drug | L-Glutamic acid | Sigma-aldrich | Sigma-aldrich: G1251 | |
| chemical compound, drug | Lipofectamin 2000 | Thermo Fisher | Thermo Fisher: 11668027 | |
| chemical compound, drug | Lipofectamin LTX | Thermo Fisher | Thermo Fisher: 15338100 | |
| chemical compound, drug | Opti-MEM I Reduced Serum Medium | Thermo Fisher | Thermo Fisher: 31985070 | |
| chemical compound, drug | Dulbecco's Modified Eagle Medium | Thermo Fisher | ThermoFirsher: 31966021 | |
| chemical compound, drug | Mg-ATP | Sigma-aldrich | Sigma-aldrich: A9187 | |
| chemical compound, drug | MgCl2 | Sigma-aldrich | Sigma-aldrich: 449172 | |
| chemical compound, drug | NaCl | Sigma-aldrich | Sigma-aldrich: S9888 | |
| chemical compound, drug | Na-GTP | Sigma-aldrich | Sigma-aldrich: G8877 | |
| chemical compound, drug | NaH2PO4 | Sigma-aldrich | Sigma-aldrich: S8282 | |
| chemical compound, drug | NPE | Synaptic Systems | SySy: 510 006 | |
| chemical compound, drug | Papain | Worthington Biochemical | Worthington Biochemical: LS003126 | |
| chemical compound, drug | Penicillin/ streptomycin | Invitrogen | Invitrogen: 15140122 | |
| chemical compound, drug | Pluronic F-127 | Thermo Fisher | Thermo Fisher: P3000MP | |
| chemical compound, drug | cg-DAG | Nadler et al., 2013 | PMID: 23720390 | |
| chemical compound, drug | caged SAG-PI(4,5)P2 | This paper | European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany. Att: Carsten Schultz (schultz@embl.de) | |
| chemical compound, drug | trypsin-inhibitor | Sigma-aldrich | Sigma-aldrich: T9253 | |
| chemical compound, drug | U73122 | Sigma-aldrich | Sigma-aldrich: U6756 | |
| chemical compound, drug | U73343 | Sigma-aldrich | Sigma-aldrich: U6881 | |
| software, algorithm | Igor Pro | Wavemetrics | ||
| ImageJ version 1.50b | Waybe Rasband, National Institute of Health, USA | |||
| SigmaPlot v. 12.3 | Systat Software Inc. | |||
| Matlab | MathWorks |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.30203.023
