1 / 31

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte. Tao Liu & al J.Neuroscience 2011. Julie JEZEQUEL Lila KHENNOUF. UE NBCM 22 Nov. 2011. Exocytosis in neurons. Small clear vesicles for neurotransmission.

giolla
Download Presentation

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Calcium Triggers Exocytosis from Two Types of Organellesin a Single Astrocyte Tao Liu & al J.Neuroscience 2011 Julie JEZEQUEL Lila KHENNOUF UE NBCM 22 Nov. 2011

  2. Exocytosis in neurons IntroductionII. Methods and results III. Conclusion IV. Perspectives Small clear vesicles for neurotransmission Large dense core vesicles for neuropeptides and hormones

  3. What about astrocytes ? Activity-dependent regulation of synaptic functions Gliotransmitters ATP D-Serine Aspartate GLUTAMATE IntroductionII. Methods and results III. Conclusion IV. Perspectives

  4. An exocytosis still under debate Different papers: • Small vesicles with VGLUT and Syb2. Ca2+ dependent exocytosis. • Lysosome (FM1-43). Ca2+ dependent exocytosis. IntroductionII. Methods and results III. Conclusion IV. Perspectives Investigation of vesicles with Ca2+ dependent exocytosis in the same astrocyte

  5. PART 1Coexistence of smallvesicles and lysosomes in astrocytes ?

  6. How to distinguishsmallvesicles and lysosomes ? Hamilton et Attwell 2010 Introduction II. Methods and results III. Conclusion IV. Perspectives Synaptobrevin 2= VAMP2 Cellubrevin= VAMP3 LAMP1

  7. General protocol Immunofluorescence detection Culture of HPC astrocytes 0-1d old Small vesiclesdetection Lysosomes detection Alexa Fluor + Ab II Alexa Fluor + Ab II Alexa Fluor + Ab II Ab I vs LAMP1 Ab I vs Syb2 Ab I vscellubrevin

  8. 1 – Do smallvesicles and lysosomes coexist in hippocampalastrocytes ? • Bothsmallvesicles and lysosomes present • in culturedastrocytes II. Methods and resultsPART 1  Figure 1 – Figure 2 – Figure 3

  9. 2 – What about freshlyisolatedastrocytes ? II. Methods and resultsPART 1  Figure 1 – Figure 2 – Figure 3  Bothsmallvesicles and lysosomes present in freshlyisolatedastrocytes

  10. An otherway to label organelles membranes FM1-43 = lipophilic marker • Used to monitor vesicle fusion in neurons • FM1-43 seems to only label lysosomes in astrocytes • (Zhang et al 2007, Li et al 2008)  Does FM1-43 only label lysosomes in culturedhippocampalastrocytes? II. Methods and resultsPART 1  Figure 1 – Figure 2 – Figure 3

  11. 3 – Does FM1-43 label smallvesicles ?  Lysotracker labels lysosomes but not smallvesicles • FM1-43 labels lysosomes • FM1-43 + EGFP-Syb2 ? • No direct evidencethat FM1-43 does not label smallvesicles ! II. Methods and resultsPART1  Figure 1 – Figure 2 – Figure 3

  12. PART 2Are both types of organelles responsible for exocytosis in astrocytes?

  13. How to investigateexocytosis in astrocytes ? Small vesicles Lysosome Mechanical stimulation Ca2+ RE Ca2+ ?

  14. How to track live fusion events ?  TIRFM =TotalInternalReflectionFluorescenceMicroscopy Classical fluorescence TIRF TOTAL REFLECTION Cell ≠ Air REFRACTION  Excites all the fluorophores present in the cell  Excites the fluorophores close to the membrane II. Methods and results PART 2  figure 4

  15. Global TIRF concept Astrocyte Non excited fluorophores Excited fluorophores Evanescent wave 100n nm Incident light beam OBJECTIVE

  16. Live imaging of smallvesicle & lysosome fusion Synapto-pHluorin Syb2 pH sensitive-GFP pH 5,5 pH 7 II. Methods and results PART 2  figure 4 EGFP-LAMP1 Ca 2+ signal • Small vesiclesexocytosis > lysosomes exocytosis

  17. Properties of smallvesicle & lysosome fusion  Fasterexocytosis for smallvesiclesthan for lysosomes II. Methods and results PART 2  figure 4  Both types of exocytosis are Ca2+- dependent

  18. Live imaging of both organelles fusion in the sameastrocyte II. Methods and results PART 2  figure 4 • Small vesicule fusion > lysosome • Fasterexocytosis for smallvesicle EGFP-LAMP1 Syb2-mOrange2

  19. PART 3Functionalrole ?

  20. Blockade of VGLUT1 inhibited glutamate release • VGLUT1 is already known to be expressed on small vesicles, but what about lysosomes ? VGLUT1 only found on small vesicles

  21. Role of VGLUT1 on glutamate release The sniffer patch technic HEK cell Astrocyte GluR-L497Y glutamate Calcium

  22. Blockade of VGLUT1 Glutamate release is decreased when VGLUT1 is blocked VGLUT1

  23. Rose began and trypan blue on calcium Importance of a control No modification of calcium by typan blue or rose bengan

  24. TeNT effects on lysosome fusion Clostridium tetani (TeNT) Syb2 (VAMP2) and Cellubrevin blocks small vesicles exocytosis

  25. Assessment of the effects on lysosomes and small vesicles TeNT has no effects on LAMP1 = syb2-pHluorin TeNT affects syb2

  26. Quantification for LAMP1 TeNT affects Syb2 TeNT does not affect LAMP1 No quantification for Syb2  Is TeNT really a tool to block small vesicle exocytosis?  Does it mean that TeNT affects small vesicles and not lysosomes?

  27. Will TeNT affect the glutamate release? First control of the TeNT effects on calcium metabolism TeNT has no effects on astrocyte calcium release

  28. Will TeNT affect the glutamate release? TeNT inhibits evoked glutamate release

  29. Conclusion & Perspectives PART 4

  30. Lysosomes Ca2+ Ca2+ Small vesicles

  31. Glutamate ? TeNT VGLUT1 How do the remain glutamate is released?

More Related