Dabral D and Coorssen JR (2019), Combined targeted Omic and Functional Assays Id...

ECB-ART-48850

Cells 2019 Apr 02;84:. doi: 10.3390/cells8040303.

Show Gene links Show Anatomy links

Combined targeted Omic and Functional Assays Identify Phospholipases A₂ that Regulate Docking/Priming in Calcium-Triggered Exocytosis.

Dabral D , Coorssen JR .

???displayArticle.abstract???
The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A₂ (PLA₂) in regulated exocytosis has long been suggested, its function or that of its metabolites-a lyso-phospholipid and a free fatty acid-remain somewhat speculative. Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA₂ were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca2+ sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA₂ and a luminal secretory PLA₂ significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.

???displayArticle.pubmedLink??? 30986994
???displayArticle.pmcLink??? PMC6523306
???displayArticle.link??? Cells

Genes referenced: LOC115919910

???attribute.lit??? ???displayArticles.show???

	Figure 1. Detection and quantification of (A) endogenous CSC FFA, n = 3 separate experiments; (B) NBD-FFA and -PA in intact CSC after 15 min labelling with NBD-PC followed by 20 min inhibitor treatments, n = 3 separate experiments; (C) total CSC neutral lipids visualized on the same TLC plate as shown in B, after charring with CuSO4. This also confirmed consistent lipid loading in each lane. Representative chromatograms showing indicated changes in lipids. (p-value, * < 0.05, < 0.005, ** < 0.0001 indicates relative difference to the control). Note: A section of chromatogram is shown in A and lanes in all chromatograms are grouped together from the same HPTLC plate following removal of lanes not associated with this study. A standard neutral lipid protocol was used to resolve the lipids [56].
	Figure 2. Effects of LY311727 (selective for sPLA2) and BEL and FKGK-11 (both selective for iPLA2) on (A) CSC Ca2+ activity curves (i.e., exocytosis in vitro); (B) fusion extent and; (C) Ca2+ sensitivity (EC50); n = 3–14. (p-value, * < 0.05, * < 0.0005, ** < 0.0001 indicates difference relative to the control).
	Figure 3. Sequence alignment of predicted urchin (A) sPLA2 and (B) 80–85 kDa iPLA2. The first row in each panel is the amino acid sequence of urchin PLA2 isozymes and the subsequent two rows are of murine and human PLA2 isozymes, respectively. Conserved amino acids are indicated with an asterisk and site # represents the amino acid residue number in the primary urchin PLA2 sequence (also considering gaps). The red arrow indicates conserved Histidine in the Ca2+ binding loop (Black box), the site at which LY311727 binds. Black arrows show conserved Histidine and Aspartic acid which form the catalytic site His-Asp dyad. Blue and red boxes indicate the caspase cleavage site and the active site serine in the GXSXG consensus sequence, respectively. The circle indicates the nucleotide binding site. Dissimilar residues with the same background colour indicate conservative substitution.
	Figure 4. Immuno-reactive spots detected by (A) sPLA2 antibody in the CV luminal fraction; (B) iPLA2 antibody in the CSC membrane fraction; and (C) iPLA2 antibody in the CV membrane fraction. cCBB stained 2D SDS-PAGE gels (left) and parallel western blots (right).
	Figure 5. (A) Time-dependent decrease in NBD-PE and no change in NBD-PC, when incubated with isolated CV luminal proteins, n = 3; (B) Detection of NBD-FFA generated from NBD-PE, when incubated with CV luminal proteins; n = 2–3. After incubations, activity was stopped by adding 0.2 µL HCl, total lipids isolated, resulting organic and aqueous phases retained, dried and resolved. (C) Bar graph summarizing the changes in NBD-PC and -PE, indicating the presence of CV luminal PLA2 with high catalytic activity for PE. (D) Effect of LY311727, increased [Ca2+]free and boiling on luminal PLA2 activity assessed by incubating isolated CV luminal proteins with NBD-PE; n = 3. (p-value, * < 0.05 indicates difference relative to the control). The chromatograms in A were resolved using a standard phospholipid protocol and in (B,D) using an established protocol for neutral lipids [56]. Note: Left panels in (A,B) show NBD fluorescence and the right panels show same section of the chromatogram after charring with CuSO4 [56]. The lanes shown in (D) are grouped together from the same chromatogram following removal of lanes not associated with this study.
	Figure 6. (A) Time-dependent changes in NBD-PC and -PE, when incubated with CV membrane fragments (n = 2–3); (B) Western blot showing an apparent complete loss of iPLA2 in trypsin treated CV (n = 3). A total of 5µg CV membrane protein per lane was resolved; (C) Time-dependent changes in the CV surface PLA2 activity in the intact CV assessed using PLA2-selective substrate PED6 [79,80] (n = 3). The loss of the fluorescence signal is consistent with the loss of PLA2 from the CV surface following ablation with trypsin. Functional assays showing the effects of trypsin treatment on CV-CV Ca2+ activity curves [45]; (D) Standard fusion assay (n = 2); (E) CV settle fusion assay (n = 3). (p-value, * < 0.05, ** < 0.005 indicates difference relative to the control). Note: The lanes in (B) are grouped together from the same western blot following removal of intervening lanes.
	Figure 7. Effects of LY311727 (selective for sPLA2) and BEL and FKGK-11 (both selective for iPLA2) on CV-CV Ca2+ activity curves, fusion extent and Ca2+ sensitivity (EC50); (A) standard fusion assay, n = 3–19; (B) settle fusion assay, n = 3–19. (p-value, * < 0.05, < 0.005, * < 0.0005, **** < 0.0001 indicates difference relative to the control.
	Figure 8. (A) Changes in the endogenous FFA in CV treated with indicated doses of LY311727, BEL and FKGK-11; n = 4–12; (B) Section of the chromatograms showing the changes in endogenous FFA. (p-value, * < 0.05, ** < 0.005 indicates difference relative to the control). A standard neutral lipid protocol was used to resolve the lipids [56].
	Figure 9. Working hypothesis: (A) Non-contacting membranes (B) Vesicle docked with the plasma membrane (PM). At basal rate, the vesicle luminal sPLA2 and membrane bound iPLA2 cleave PE and PC in the inner and outer monolayer, respectively; PM bound iPLA2 also cleaves PC in the inner monolayer of the PM. This causes a local increase in LPC at the membrane contact site that is balanced by intermittent LPE production at the adjacent inner membrane via transient sPLA2 activity. This mechanism may thus act as a native fusion ‘brake’ and the released FFA (often Arachidonic acid) may support trans SNARE complex formation to ensure and maintain efficient priming/docking. (adapted from [29] with permission).

References [+] :

Abbineni, Application of High-Throughput Assays to Examine Phospho-Modulation of the Late Steps of Regulated Exocytosis. 2017, Pubmed