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First published online 6 June 2006
doi: 10.1242/jcs.03007

Journal of Cell Science 119, 2688-2694 (2006)
Published by The Company of Biologists 2006
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Sphingomyelin-enriched microdomains define the efficiency of native Ca2+-triggered membrane fusion

Tatiana Rogasevskaia and Jens R. Coorssen*

Departments of Physiology and Biophysics, Biochemistry and Molecular Biology, and Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Faculty of Medicine, Calgary, AB, T2N 4N1, Canada


Figure 1
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  		Fig. 1. The dose-dependent effects of SMase on CV fusion. (a) Ca2+-activity curves at 0 (n=15), 1 (n=7), 10 (n=15) and 20 U/ml SMase (n=5). Vertical dotted lines indicate the EC50 of each curve (P<0.05). (b) Kinetics of CV fusion triggered with 78.2±5.4 µM [Ca2+]free after treatment with 0, 1 and 20 U/ml SMase (n=5); symbols are as for a. (c) Changes in CV sphingomyelin relative to untreated controls (n=3) after exposure to 1, 5 and 10 U/ml of SMase. *Significant difference from control (P<0.05); **significant difference from control and previous conditions (P<0.001).

 

Figure 2
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  		Fig. 2. Effects of SMase and subsequent CHOL loading (using CHOL-loaded hpßcd) on CV fusion kinetics triggered with 90.2±7.3 µM [Ca2+]free.

 

Figure 3
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  		Fig. 3. Effects of SMase and mßcd on CV fusion and Cer concentration. (a) Ca2+-activity curves following treatments with mßcd (2 mM) before and after CV exposure to SMase (10 U/ml). Vertical dotted lines indicate the EC50 of each curve. (b) Kinetics of CV fusion triggered with 82.5±4.1 µM [Ca2+]free after treatment with mßcd and/or SMase, as indicated (n=5); symbols are as for a. (c) Total CV ceramide assayed after SMase treatments ± exposure to mßcd (mbcd; 2 mM), as indicated (n=2). *Significant difference from control and the mßcd + SMase treatment (P<0.01).

 

Figure 4
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  		Fig. 4. Effects of lysenin on CV fusion. (a) Ca2+-activity curves following treatments with lysenin, before (n=8) and after CV exposure to SMase (10 U/ml) (n=4). Vertical dotted lines indicate the EC50 of each curve. (b) Kinetics of CV fusion triggered with 86.9±5.8 µM [Ca2+]free after treatment with 0 and 0.6 nM lysenin (n=8).

 




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