Skip to main content
. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Exp Cell Res. 2011 Jan 20;317(6):691–705. doi: 10.1016/j.yexcr.2011.01.008

Figure 3. Comparison between the enzyme and channel activities and the contribution to lysosomal exocytosis of WT-MLN1 and SL-MLN1 mutant.

Figure 3

In (a) the Bodipy FL C11-PC probe was used to assess the phospholipase activities of WT-MLN1 (closed triangles), SL-MLN1 (open squares), and the control (H2O-injected oocytes) (+/−SEM, n=12). The activity was estimated in LE/L-containing oocyte extracts for each of these groups by measuring the increase in fluorescence intensity units (FIU) several days after injection. In (b) the K+, Na+, and Ca2+ conductances, expressed in picosiemens (pS), of WT-MLN1 (open bars) and SL-MLN1 (dark bars) are compared. The unitary conductances were 81+/−6.4 pS (+/−SEM, n=8) for K+, 77.9+/−7.8 pS (n=8) for Na+, and 30.4+/−5.5 pS (n=8). Single channel traces were taken on day 2 (c) and day 6 (d) after injection of oocytes with WT-MLN1 cRNA (top traces) or SL-MLN1 cRNA (bottom traces). The traces were taken at −90 mV in the presence of 100 mM KCl in the pipette solution. The increase in open state probability (nPo) several days after injection (e) is similar in WT-MLN1-(open triangles) and SL-MLN1-expressing oocytes (closed circles) (+/−SEM, n=12). The lysosomal exocytosis was assessed by determining the amount of N-acetyl-beta-D-glucosaminidase (NAG) (+/−SEM, n=6) released into the bath solution from untreated whole oocytes (−) and from oocytes treated for 30 min with 10 μM ionomycin (+), as shown in (f). The three pairs of bars are marked underneath from left to right for H2O-injected oocytes (Ctrl), or oocytes expressing either WT-MLN1 (WT) or SL-MLN1 (SL). The released amount of NAG is expressed as % of its total content in the cells, as explained in Methods. (g-h). Effects of phospholipase inhibitors on the enzyme activity and on the extrusion of TVS from oocytes expressing WT-MLN1. Panel (g) shows the changes in PLA activity of WT-MLN1 in LE/L-containing oocyte extracts (estimated by measuring the fluorescence of the Bodipy FL C11-PC probe) mediated by the PLA2 inhibitors, aristolochic acid (Aris, 40 μM) and bromophenacyl bromide (BPB, 40 μM), as well as by the inhibitor of phosphatidylinositol-specific PLC, U73122 (2 μM), and the inhibitor of phosphatidylcholine-specific PLC and PLD, D609 (40 μM) (+/−SEM, n=10). Panel (h) shows that BPB (open triangles) and ArisA (closed squares) markedly reduce the percentage of whole oocytes extruding TVS, while the inhibitors of PLC and PLD (crosses and open circles) do not show significant changes in comparison to the untreated oocytes (Untr.) (closed circles)(+/−SEM, n=6).