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Published online 11 July 2000. doi:10.1083/jcb.150.1.155
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© The Rockefeller University Press, 0021-9525/2000/7/155/ $5.00
The Journal of Cell Biology, Volume 150, Number 1, July 10, 2000 155-164

Original Article

Sphingomyelin Hydrolysis to Ceramide during the Execution Phase of Apoptosis Results from Phospholipid Scrambling and Alters Cell-surface Morphology

Annemiek D. Teppera, Paula Ruursa, Therese Wiedmerb, Peter J. Simsb, Jannie Borsta, and Wim J. van Blitterswijka
a Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
b Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037

Correspondence to: Wim J. van Blitterswijk, Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel:31-20-512-1976 Fax:31-20-512-1989 E-mail:wblit{at}nki.nl.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]–sphingosylphosphorylcholine (C6-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca2+ ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-ß-cyclodextrin, which is indicative of a loss of tight SM–cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C6- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Key Words: CD95, cholesterol, PS exposure, DNA damage, blebbing


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