Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro
A. Prinetti et al., Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro, J BIOL CHEM, 276(24), 2001, pp. 21136-21145
In the present paper, we report on the properties of sphingolipid-enriched
domains of rat cerebellar granule cells in culture at different stages of n
euronal development. The major lipid components of these domains were glyce
rophospholipids and cholesterol, Glycerophospholipids were 45-75% and chole
sterol 15-45% of total lipids of the domains. This corresponded to 5-17% of
total cell glycerophospholipids and 15-45% of total cell cholesterol, Phos
phatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all t
he glycerophospholipids associated with these domains. Consequently, the pa
lmitoyl residue was significantly enriched in the domains. The surface occu
pied by these structures increased during development. 40-70% of cell sphin
golipids segregated in sphingolipid-enriched membrane domains, with the max
imum ganglioside density in fully differentiated neurons. A high content of
ceramide was found in the domains of aging neurons. Then, the sphingolipid
/glycerophospholipid molar ratio was more than doubled during the initial s
tage of development, whereas the cholesterol/glycerophospholipid molar rati
o gradually decreased during in vitro differentiation. Phosphorylated phosp
hoinositides, which were scant in the domains of undifferentiated cells, dr
amatically increased during differentiation and aging in culture. Proteins
were minor components of the domains (0.1-2.8% of all domain components). P
hosphotyrosine-containing proteins were selectively recovered in the sphing
olipid-enriched domain. Among these, Src family protein-tyrosine kinases, k
nown to participate to the process of neuronal differentiation, were associ
ated with the sphingolipid-enriched domains in a way specific for the type
of kinase and for the developmental stage of the cell. Proteins belonging t
o other signaling pathways, such as phosphoinositide 3-kinase and its downs
tream target, Akt, were not associated with the domains.