Konfokal mikroskopi til måling af tre tilstande af fusionsporedynamik i binyrekromaffinceller
Summary
Denne protokol beskriver en konfokal billeddannelsesteknik til påvisning af tre fusionstilstande i bovin adrenal kromaffinceller. Disse fusionstilstande omfatter 1) tæt fusion (også kaldet kiss-and-run), der involverer fusionsporeåbning og lukning, 2) opholdsfusion, der involverer fusionsporeåbning og vedligeholdelse af den åbne pore, og 3) krympefusion, der involverer smeltet vesikelkrympning.
Abstract
Dynamisk fusion pore åbning og lukning medierer exocytose og endocytose og bestemmer deres kinetik. Her demonstreres det i detaljer, hvordan konfokal mikroskopi blev brugt i kombination med patch-clamp-optagelse til at detektere tre fusionstilstande i primære dyrkningskrumbarkhormonceller. De tre fusionstilstande omfatter 1) tæt fusion (også kaldet kiss-and-run), der involverer fusionsporeåbning og lukning, 2) opholdsfusion, der involverer fusionsporeåbning og vedligeholdelse af den åbne pore, og 3) krympefusion, der involverer krympning af den fusionsgenererede Ω-formprofil, indtil den smelter helt sammen ved plasmamembranen.
For at detektere disse fusionstilstande blev plasmamembranen mærket ved overekspression af mNeonGreen fastgjort med PH-domænet for phospholipase C δ (PH-mNG), som binder til phosphatidylinositol-4,5-bisphosphat (PtdIns (4,5) P2) ved plasmamembranens cytosolvendte folder; vesikler blev fyldt med den fluorescerende falske neurotransmitter FFN511 for at detektere frigivelse af vesikulært indhold; og Atto 655 blev inkluderet i badeopløsningen for at detektere fusionsporelukning. Disse tre fluorescerende sonder blev afbildet samtidigt ved ~ 20-90 ms pr. Ramme i levende kromaffinceller for at detektere fusionsporeåbning, indholdsfrigivelse, fusionsporelukning og fusion af vesikelstørrelsesændringer. Analysemetoden er beskrevet for at skelne mellem tre fusionstilstande og disse fluorescensmålinger. Metoden beskrevet her kan i princippet gælde for mange sekretoriske celler ud over kromaffinceller.
Introduction
Membranfusion formidler mange biologiske funktioner, herunder synaptisk transmission, blodsukkerhomeostase, immunrespons og viral indgang 1,2,3. Exocytose, der involverer vesikelfusion ved plasmamembranen, frigiver neurotransmittere og hormoner for at opnå mange vigtige funktioner, såsom neuronale netværksaktiviteter. Fusion åbner en pore for at frigive vesikulært indhold, hvorefter poren kan lukke for at hente fusionsvesikel, som kaldes kiss-and-run 1,4. Både irreversibel og reversibel fusionsporeåbning kan måles med celletilsluttede kapacitansoptagelser kombineret med fusionsporekonduktansoptagelser af enkelt vesikelfusion.
Dette fortolkes ofte som udtryk for fuldkollapsfusion, der involverer udvidelse af fusionen indtil udfladning af smelteversilen, og kys-og-løb, der involverer fusionsporeåbning og lukning, henholdsvis 5,6,7,8,9,10,11,12,13 . Nylige konfokale og stimulerede emissionsudtømning (STED) billeddannelsesundersøgelser i kromaffinceller observerede direkte fusionsporeåbning og lukning (kiss-and-run, også kaldet tæt fusion), fusionsporeåbning, der opretholder en Ω form med en åben pore i lang tid, betegnet opholdsfusion og krympning af fusionsveklen, indtil den er fuldstændig fusioneret med plasmamembranen, som erstatter fuldkollapsfusionsfusion til fusion af vesikler med plasmamembranen4, 8,14,15,16,17.
I neuroner er fusionsporeåbning og -lukning blevet detekteret med billeddannelse, der viser frigivelsen af kvanteprikker forudindlæst i vesikler, der er større end fusionsporen, og med fusionsporekonduktansmålinger ved frigivelsesfladen af nerveterminaler 5,18,19. Adrenal kromaffinceller anvendes i vid udstrækning som en model til undersøgelse af exo- og endocytose20,21. Selvom kromaffinceller indeholder store tætte kernevesikler, mens synapser indeholder små synaptiske vesikler, er exocytose- og endocytoseproteinerne i kromaffinceller og synapser ret analoge 10,11,12,20,21,22,23.
Her beskrives en metode til måling af disse tre fusionstilstande ved hjælp af en konfokal billeddannelsesmetode kombineret med elektrofysiologi i bovin adrenalkromaffinceller (figur 1). Denne metode involverer indlæsning af fluorescerende falske neurotransmittere (FFN511) i vesikler for at detektere exocytose; tilsætning af Atto 655 (A655) i badeopløsningen for at fylde den fusionsgenererede Ω-formprofil og mærkning af plasmamembranen med PH-domænet for phospholipase C δ (PH), som binder til PtdIns (4,5) P2 ved plasmamembranen 8,15,24. Fusionsporedynamik kan detekteres gennem ændringer i forskellige fluorescerende intensiteter. Selvom det er beskrevet for chromaffinceller, kan princippet om denne metode, der er beskrevet her, anvendes bredt på mange sekretoriske celler langt ud over kromaffinceller.
Protocol
Representative Results
Discussion
En konfokal mikroskopisk billeddannelsesmetode er beskrevet for at detektere dynamikken i fusionspore og transmitterfrigivelse samt tre fusionstilstande, tæt fusion, opholdsfusion og krympefusion i bovin adrenalchromffinceller 4,24. En elektrofysiologisk metode til at depolarisere cellen og derved fremkalde exo- og endocytose er beskrevet. Systematisk konfokal billedbehandling giver information om forskellige former for poreadfærd for fusions- og fissionshænde…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker NINDS Intramural Research Programs (ZIA NS003009-13 og ZIA NS003105-08) for at støtte dette arbejde.
Materials
| Adenosine 5'-triphosphate magnesium salt | Sigma | A9187-500MG | ATP for preparing internal solution |
| Atto 655 | ATTO-TEC GmbH | AD 655-21 | Atto dye to label bath solution |
| Basic Nucleofector for Primary Neurons | Lonza | VSPI-1003 | Electroporation transfection buffer along with kit |
| Boroscilicate capillary glass pipette | Warner Instruments | 64-0795 | Standard wall with filament OD=2.0 mm ID=1.16 mm Length=7.5 cm |
| Bovine serum albumin | Sigma | A2153-50G | Reagent for gland digestion |
| Calcium Chloride 2 M | Quality Biological | 351-130-721 | Reagent for preparing bath solution |
| Cell Strainers, 100 µm | Falcon | 352360 | Material for filtering chromaffin cell suspension |
| Cesium hydroxide solution | Sigma | 232041 | Reagent for preparing internal solution and Cs-glutamate/Cs-EGTA stock buffer |
| Collagenase P | Sigma | 1.1214E+10 | Enzyme for gland digestion |
| Coverslip | Neuvitro | GG-14-Laminin | GG-14-Laminin, 14 mm dia.#1 thick 60 pieces Laminin coated German coverslips |
| D-(+)-Glucose | Sigma | G8270-1KG | Reagent for preparing Locke’s solution and bath solution |
| DMEM | ThermoFisher Scientific | 11885092 | Reagent for preparing culture medium |
| EGTA | Sigma | 324626-25GM | Reagent for preparing Cs-EGTA stock buffer for bath solution |
| Electroporation and Nucleofector | Amaxa Biosystems | Nucleofector II | Transfect plasmids into cells |
| Fetal bovine serum | ThermoFisher Scientific | 10082147 | Reagent for preparing culture medium |
| FFN511 | Abcam | ab120331 | Fluorescent false neurotransmitter to label vesicles |
| Guanosine 5'-triphosphate sodium salt hydrate | Sigma | G8877-250MG | GTP for preparing internal solution |
| HEPES | Sigma | H3375-500G | Reagent for preparing Locke’s solution |
| Igor Pro | WaveMetrics | Igor pro | Software for patch-clamp analysis and imaging data presentation |
| Leica Application Suite X software | Leica | LAS X software | Confocal software for imaging data collection and analysis |
| Leica TCS SP5 Confocal Laser Scanning Microscope | Leica | Leica TCS SP5 | Confocal microscope for imaging data collection |
| L-Glutamic acid | Sigma | 49449-100G | Reagent for preparing Cs-glutamate stock buffer for bath solution |
| Lock-in amplifier | Heka | Lock-in | Software for capacitance recording |
| Magnesium Chloride 1 M | Quality Biological | 351-033-721EA | Reagent for preparing internal solution and bath solution |
| Metallized Hemacytometer Hausser Bright-Line | Hausser Scientific | 3120 | Counting chamber |
| Microforge | Narishige | MF-830 | Polish pipettes to enhance the formation and stability of giga-ohm seals |
| Millex-GP Syringe Filter Unit, 0.22 µm | Millipore | SLGPR33RB | Material for glands wash and digestion |
| mNG(mNeonGreen) | Allele Biotechnology | ABP-FP-MNEONSB | Template for PH-mNeonGreen construction |
| Nylon mesh filtering screen 100 micron | EIKO filtering co | 03-100/32 | Material for filtering medulla suspension |
| Patch clamp EPC-10 | Heka | EPC-10 | Amplifier for patch-clamp data collection |
| PH-EGFP | Addgene | Plasmid #51407 | Backbone for PH-mNeonGreen construction |
| Pipette puller | Sutter Instrument | P-97 | Make pipettes for patch-clamp recording |
| Potassium Chloride | Sigma | P5404-500G | Reagent for preparing Locke’s solution and bath solution |
| Pulse software | Heka | Pulse | Software for patch-clamp data collection |
| Recording chamber | Warner Instruments | 64-1943/QR-40LP | coverslip chamber, apply patch-clamp pipette on live cells |
| Sodium chloride | Sigma | S7653-1KG | Reagent for preparing Locke’s solution, bath solution and internal solution |
| Sodium hydroxide | Sigma | S5881-500G | Reagent for preparing Locke’s solution |
| Sodium phosphate dibasic | Sigma | S0876-500G | Reagent for preparing Locke’s solution |
| Sodium phosphate monobasic | Sigma | S8282-500G | Reagent for preparing Locke’s solution |
| Stirring hot plate | Barnsted/Thermolyne | type 10100 | Heater for pipette coating with wax |
| Syringe, 30 mL | Becton Dickinson | 302832 | Material for glands wash and digestion |
| Tetraethylammonium chloride | Sigma | T2265-100G | TEA for preparing bath solution |
| Trypsin inhibitor | Sigma | T9253-5G | Reagent for gland digestion |
| Type F Immersion liquid | Leica | 195371-10-9 | Leica confocal mounting oil |
Referências
- Wu, L. G., Hamid, E., Shin, W., Chiang, H. C. Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annual Review of Physiology. 76, 301-331 (2014).
- Chang, C. W., Chiang, C. W., Jackson, M. B. Fusion pores and their control of neurotransmitter and hormone release. Journal of General Physiology. 149 (3), 301-322 (2017).
- Harrison, S. C. Viral membrane fusion. Nature Structural & Molecular Biology. 15 (7), 690-698 (2008).
- Zhao, W. D., et al. Hemi-fused structure mediates and controls fusion and fission in live cells. Nature. 534 (7608), 548-552 (2016).
- Klyachko, V. A., Jackson, M. B. Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals. Nature. 418 (6893), 89-92 (2002).
- Albillos, A., et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature. 389 (6650), 509-512 (1997).
- He, L., Wu, X. S., Mohan, R., Wu, L. G. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature. 444 (7115), 102-105 (2006).
- Chiang, H. C., et al. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles. Nature Communications. 5, 3356 (2014).
- Sharma, S., Lindau, M. The fusion pore, 60 years after the first cartoon. Federation of European Biochemical Societies Letters. 592 (21), 3542-3562 (2018).
- Jorgacevski, J., et al. Munc18-1 tuning of vesicle merger and fusion pore properties. Journal of Neuroscience. 31 (24), 9055-9066 (2011).
- Rituper, B., et al. Vesicle cholesterol controls exocytotic fusion pore. Cell Calcium. 101, 102503 (2021).
- Gucek, A., et al. Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state. Cellular and Molecular Life Sciences. 73 (19), 3719-3731 (2016).
- Grabner, C. P., Moser, T. Individual synaptic vesicles mediate stimulated exocytosis from cochlear inner hair cells. Proceedings of the National Academy of Sciences of the United States of America. 115 (50), 12811-12816 (2018).
- Wen, P. J., et al. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane. Nature Communications. 7, 12604 (2016).
- Shin, W., et al. Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis. Cell. 173 (4), 934-945 (2018).
- Shin, W., et al. Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents. Cell Reports. 30 (2), 421-431 (2020).
- Ge, L., Shin, W., Wu, L. -. G. Visualizing sequential compound fusion and kiss-and-run in live excitable cells. bioRxiv. , (2021).
- Zhang, Q., Li, Y., Tsien, R. W. The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science. 323 (5920), 1448-1453 (2009).
- He, L., Wu, X. S., Mohan, R., Wu, L. G. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature. 444 (7115), 102-105 (2006).
- Androutsellis-Theotokis, A., Rubin de Celis, M. F., Ehrhart-Bornstein, M., Bornstein, S. R. Common features between chromaffin and neural progenitor cells. Molecular Psychiatry. 17 (4), 351 (2012).
- Bornstein, S. R., et al. Chromaffin cells: the peripheral brain. Molecular Psychiatry. 17 (4), 354-358 (2012).
- Park, Y., Kim, K. T. Short-term plasticity of small synaptic vesicle (SSV) and large dense-core vesicle (LDCV) exocytosis. Cellular Signalling. 21 (10), 1465-1470 (2009).
- Thahouly, T., Niedergang, F., Vitale, N., Gasman, S. Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion. Exocytosis and Endocytosis. Methods in Molecular Biology. 2233, (2021).
- Shin, W., et al. Preformed Omega-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells. Neuron. 109 (19), 3119-3134 (2021).
- O’Connor, D. T., et al. Primary culture of bovine chromaffin cells. Nature Protocols. 2 (5), 1248-1253 (2007).
- Wu, X. S., et al. Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells. Biophysical Journal. 113 (11), 2406-2414 (2017).
- Revelo, N. H., et al. A new probe for super-resolution imaging of membranes elucidates trafficking pathways. Journal of Cell Biology. 205 (4), 591-606 (2014).
- Gao, J., Liao, J., Yang, G. -. Y. CAAX-box protein, prenylation process and carcinogenesis. American journal of translational research. 1 (3), 312-325 (2009).
- Schermelleh, L., et al. Super-resolution microscopy demystified. Nature Cell Biology. 21 (1), 72-84 (2019).
- Ceccarelli, B., Hurlbut, W. P., Mauro, A. Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. Journal of Cell Biology. 54 (1), 30-38 (1972).
- Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods. 3 (10), 793-795 (2006).
- Betzig, E., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313 (5793), 1642-1645 (2006).
- Fish, K. N. Total internal reflection fluorescence (TIRF) microscopy. Current Protocols in Cytometry. , 18 (2009).
- Schmidt, R., et al. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nature Communications. 12 (1), 1478 (2021).
