From the Study Center for the Functional Biochemistry of Brain Lipids, Department of Medical Chemistry and Biochemistry-Laboratorio Interdisciplinare Tecnologie Avanzate, Medical School, University of Milan, Segrate, Italy 20090
Received for publication, November 27, 2000, and in revised form, March 13, 2001
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ABSTRACT |
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In the present paper, we report
on the properties of sphingolipid-enriched domains of rat cerebellar
granule cells in culture at different stages of neuronal development.
The major lipid components of these domains were glycerophospholipids
and cholesterol. Glycerophospholipids were 45-75% and cholesterol
15-45% of total lipids of the domains. This corresponded to 5-17%
of total cell glycerophospholipids and 15-45% of total cell
cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all the
glycerophospholipids associated with these domains. Consequently, the
palmitoyl residue was significantly enriched in the domains. The
surface occupied by these structures increased during development.
40-70% of cell sphingolipids segregated in sphingolipid-enriched
membrane domains, with the maximum 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 stage of
development, whereas the cholesterol/glycerophospholipid molar ratio
gradually decreased during in vitro differentiation.
Phosphorylated phosphoinositides, which were scant in the domains of
undifferentiated cells, dramatically increased during differentiation
and aging in culture. Proteins were minor components of the domains
(0.1-2.8% of all domain components). Phosphotyrosine-containing
proteins were selectively recovered in the sphingolipid-enriched
domain. Among these, Src family protein-tyrosine kinases, known to
participate to the process of neuronal differentiation, were associated
with the sphingolipid-enriched domains in a way specific for the type
of kinase and for the developmental stage of the cell. Proteins
belonging to other signaling pathways, such as phosphoinositide
3-kinase and its downstream target, Akt, were not associated with the domains.
Neuronal development involves many different spatially
and temporally regulated events (reviewed in Refs. 1-4), including cell migration (which implies changes in the adhesion properties of the
neuronal cell and in its interactions with the changing local
environment and other cells), and deep morphological and biochemical
changes leading to the processes of neuritogenesis and synaptogenesis,
and to the complex spatial organization which is typical of the nervous
tissue. In all these developmental events, cell surface properties,
determined by membrane components, are of primary importance. Lipids
are responsible for the physico-chemical properties of the membrane
itself, largely determining its asymmetry, fluidity and plasticity
features, and its organization in domains. Moreover, many membrane
lipids play more specific functional roles, and are in many different
ways directly involved in the machinery devoted to the control of
information in the neuronal cell. Inositol phospholipids (5),
sphingolipids (6, 7), and plasmalogens (8) are precursors of bioactive
molecules. Glycosphingolipids, asymmetrically located in the outer
leaflet of the membrane bilayer, are of primary importance as receptors
and cell surface antigens (9). A particular function of lipids at the
cell surface is linked to their ability to interact with membrane
proteins, such as receptors or ion channels (6, 10, 11), and to
modulate the functional activity of the protein itself. Some membrane
lipids undergo segregation, creating a lipid phase distinct from the bulk phase of the membrane. Recent findings indicate that a particular set of lipids and proteins are segregated together within the plasma
membrane, forming functional units that are involved in signal
transduction processes (12, 13). Spontaneous segregation of membrane
sphingolipids seems to be the driving force of these structures, hence
termed sphingolipid-enriched membrane domains. A functional role for
these domains is particularly well documented in neural cells (reviewed
in Ref. 14), where the specific interaction of sphingolipids with
protein kinases of the Src family is particularly relevant. The
nonreceptor protein-tyrosine kinases belonging to this family
seem to be involved in signaling pathways that regulate the function of
postmitotic, terminally differentiated cells. They regulate multiple
cell functions, including cytoskeletal organization, cell-substrate,
and cell-cell adhesion, and cell-cell communication (15, 16). In the
nervous system, many lines of evidence indicate that they are involved
in the process of neuronal differentiation, suggesting a tight link
between Src family kinases and neurite extension and guidance. In the
developing cerebellum, the expression of c-Src coincides with the onset
of neuronal differentiation (17). The expression and activation of
c-Src and Lyn are increased during differentiation (18-22), and
postmitotic neurons from the central nervous system express high levels
of structurally distinct forms of c-Src and Fyn (15, 23). c-Src and Fyn
are required in the response to different neural cell adhesion
molecules in cerebellar neurons (24, 25), participating in the control
of neurite extension. c-Src, Fyn, and Yes are enriched in growth cones
of neurons and neuroblastoma cells as a complex with other proteins
(26). c-Src and Fyn also have a role in the mature synapse, related to
the induction of long-term potentiation (27, 28). A close association
between these proteins and sphingolipid-enriched domains has been
demonstrated in many cell types, including neurons (29-32). Moreover,
several GPI1-anchored
proteins, which are usually associated with the sphingolipid-enriched domains, have been implicated in signal transduction mediated by Src
family tyrosine kinases. In the present paper, we analyze the changes
in lipid composition, turnover, and membrane organization in cerebellar
neurons at different stages of development in vitro. The
data we obtained clearly indicate a strict correlation between the
functional status of the neuronal cell and the level of lipid and
protein organization within sphingolipid-enriched membrane domains.
Materials--
Commercial chemicals were the purest available,
common solvents were distilled before use, and water was doubly
distilled in a glass apparatus. Trypsin, crystalline bovine serum
albumin, and all the reagents for cell culture and the cholesterol
assay kit were from Sigma Chemical Co., except for basal modified
Eagle's medium and fetal calf serum, which were purchased from Flow
Laboratories. Anti-phosphotyrosine mouse monoclonal IgG2b
antibody, anti-c-Src goat polyclonal IgG (N-16), anti-c-Src (SRC 2),
anti-Lyn, anti-Fyn, anti-Akt1/2, anti-PI3K (p110) rabbit
polyclonal IgG antibodies, horseradish peroxidase-conjugated secondary
antibodies, and Protein A/G PLUS-agarose were from Santa Cruz
Biotechnology. Sphingosine was prepared from cerebroside (33). Standard
sphingolipids and glycerophospholipids were extracted from rat brain,
purified, and characterized (34). [1-3H]Sphingosine was
prepared by specific chemical oxidation of the primary hydroxyl group
of sphingosine followed by reduction with sodium
boro[3H]hydride (35) (radiochemical purity over 98%;
specific radioactivity 2 Ci/mmol). [32P]Orthophosphate
(carrier-free) and [35S]methionine (specific
radioactivity 1175 Ci/mmol) were purchased from Amersham Pharmacia
Biotech and PerkinElmer Life Sciences, respectively.
3H-Labeled lipids were extracted from
[1-3H]sphingosine-fed cells, purified, characterized, and
used as chromatographic standards.
Cell Culture--
Granule cells, obtained from the cerebella of
8-day-old Harlan Sprague-Dawley rats, were prepared and cultured as
described previously (36-38). The cells were plated in 100-mm dishes
at a density of 9 × 106 cells/dish and cultured with
10 ml of basal modified Eagle's medium containing 10% fetal calf
serum for up to 17 days. After the 8th day in culture, the medium was
supplemented with 1 mg/ml glucose. Cell viability was assessed by the
Trypan blue exclusion method. Primary cultures from 8-day-old rat
cerebellum are greatly enriched in neurons. Contamination by
non-neuronal cells (mainly astrocytes), as determined by immunological
and morphological criteria, is below 5% (37, 38). The replication of
non-neuronal cells was prevented by adding 10 µM cytosine
arabinoside to the culture medium. In these cultures, about 90% of the
cells are immature excitatory granule cells. The cultures also contain
about 5% Analysis of Endogenous Lipids--
Cells (4-7 × 107) at the 2nd, 8th, and 17th day in culture were
harvested and lyophilized. Lipids were extracted with
chloroform/methanol 2:1 (v/v), and the total lipid extract was
subjected to a two-phase partitioning as previously described (41),
resulting in the separation of an aqueous phase containing gangliosides
and in an organic phase containing all other lipids. The ganglioside content was determined in the aqueous phase as lipid-bound sialic acid
by the method of Svennerholm (42). The phospholipid content was
determined in the organic phases as phosphate after perchloric acid
digestion by the method of Bartlett (43). Gangliosides from the aqueous
phases and phospholipids, cholesterol, and ceramide from the organic
phases were separated by HPTLC as described below. Identification of
lipids after separation was assessed by comigration with standard
lipids and confirmed by their susceptibility to enzymatic and chemical
treatments as previously described (44). After chromatographic
separation, compounds were chemically detected and their amounts were
determined by densitometry as described below.
The mass lipid composition of the sphingolipid-enriched membrane
fractions was calculated on the basis of the endogenous lipid content
in the cell homogenate and the percent distribution of the
radioactivity associated with each lipid species in the fraction, as
described below.
Treatments of Cell Cultures with [3H]Sphingosine or
[35S]Methionine or
[32P]Orthophosphate--
Cells at the day of preparation
(0 day in culture), at the 6th and at the 15th day in culture, were
incubated in the presence of 3 × 10 Sucrose Gradient Centrifugation--
After metabolic
radiolabeling with [1-3H]sphingosine, or
[35S]methionine, or [32P]orthophosphate, at
the 2nd, 8th, and 17th days in culture cells were subjected to
ultracentrifugation on discontinuous sucrose gradients as previously
described (44). Briefly, cells were harvested, lysed in lysis buffer
(1% Triton X-100, 10 mM Tris buffer, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 75 milliunits/ml aprotinin, 5-8 × 107
cells/ml) and Dounce homogenized (10 strokes, tight). Cell lysate was
centrifuged (5 min, 1300 × g) to remove nuclei and
cellular debris. The postnuclear fraction was mixed with an equal
volume of 85% sucrose (w/v) in 10 mM Tris buffer (pH 7.5),
150 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4, placed at the bottom of a discontinuous sucrose concentration gradient (30-5%) in the same buffer, and centrifuged (17 h, 200,000 × g) at 4 °C. After
ultracentrifugation, eleven fractions were collected starting from the
top of the tube. The light-scattering band located at the interface
between 5 and 30% sucrose and corresponding to fraction 5 was regarded
as the sphingolipid-enriched membrane fraction (SEMF) (44). The entire procedure was performed at 0-4 °C in ice immersion.
Analysis of Radioactive Lipids--
The cell lysate, postnuclear
supernatant, and sucrose gradient fractions obtained after cell
metabolic radiolabeling were analyzed to determine the content of
radiolabeled lipids. Samples were dialyzed and lyophilized, and lipids
were extracted with chloroform/methanol 2:1 (v/v) (41). The total lipid
extract was analyzed by HPTLC as described below, followed by
radioactivity imaging for quantification of radioactivity.
Identification of lipids separated by HPTLC was accomplished by
comigration with standard lipids and confirmed by susceptibility of
compounds to following enzymatic and chemical treatments (44). A sample
of the lipid extract was treated at 37 °C for 2 h, in 50 µl
of water, in the presence of 1 milliunit of Vibrio cholerae
sialidase, to yield GM1. Sphingomyelin, phosphatidylethanolamine, and
phosphatidylcholine were purified according to the HPTLC-blotting
technique previously reported (48): they were separated by HPTLC,
identified by spraying with primulin, blotted to PVDF membrane where
the corresponding bands were cut and subjected to elution.
Sphingomyelin was treated at 37 °C overnight in 30 µl of 100 mM Tris-HCl, pH 7.4, 0.5 mM MgCl2,
0.05% sodium deoxycholate, in the presence of 11 milliunits of
Bacillus cereus sphingomyelinase, to yield ceramide;
phosphatidylethanolamine was characterized following its degradation
under alkaline conditions. The enzymatic or chemical reaction mixtures
were separated by HPTLC, and the reaction products were identified by
chromatographic comparison with standard lipids.
Thin Layer Chromatography--
3H-Lipids were
separated by monodimensional HPTLC carried out with the solvent systems
chloroform/methanol/0.2% aqueous CaCl2, 50:42:11 or
55:45:10 (v/v). Endogenous gangliosides were separated by bidimensional
HPTLC using the solvent system chloroform/methanol/0.2% aqueous
CaCl2, 50:42:11 for the 1st and 2nd run, with intermediate exposure to ammonia vapors as previously described (49), to allow the
detection of alkali-labile species.
Endogenous phospholipids or [32P]lipids were separated by
mono- or bidimensional HPTLC using the following solvent systems and conditions (44). Monodimensional HPTLC: (a)
chloroform/methanol/acetic acid/water, 30:20:2:1 (v/v), for the
separation of PE, phosphatidylinositol, PS, PC, and sphingomyelin;
(b) chloroform/acetone/methanol/acetic acid/water,
40:15:13:12:8 (v/v), to specifically analyze the content of
phosphatidylinositol phosphates; the separation was carried out on
potassium oxalate-impregnated HPTLC plates. Bidimensional HPTLC:
(a) first run, chloroform/methanol/acetic acid/water,
30:20:2:1 (v/v); second run, chloroform/methanol/acetone/acetic
acid/water, 10:2:4:2:1 (v/v), with intermediate exposure to HCl vapors
for 15 min; this bidimensional HPTLC system allowed the analysis of plasmalogens in the glycerophospholipid mixture.
Ganglioside and glycerophospholipid species were quantified after
separation on HPTLC followed by specific detection with a
p-dimethylaminobenzaldehyde reagent (50), or a molybdate
reagent (51), respectively. The relative amounts of each ganglioside or
of each phospholipid were determined by densitometry, and their mass
content was calculated on the basis of the percent distribution and
total ganglioside and phospholipid content, determined as described above.
Cholesterol and ceramide were separated by monodimensional HPTLC using
the solvent systems hexane/diethylether/acetic acid, 80:20:1, and
hexane/chloroform/acetone/acetic acid, 10:35:10:1, respectively.
Cholesterol and ceramide were quantified after separation on HPTLC
followed by visualization with 15% concentrated sulfuric acid in
1-butanol. The quantities of cholesterol and ceramide were determined
by densitometry and comparison with 0.1-2 µg of standard compounds
using the Molecular Analyst program (Bio-Rad Laboratories). In the case
of cholesterol, the S.D. of the procedure was ±20% in the range of
0.1-0.5 µg, and ±10% in the range of 1-2 µg. These values were
similar to those already reported for analytical methods based on HPTLC
separation and charring of cholesterol (52). In preliminary
experiments, we obtained similar results on the cell cholesterol
content using the TLC-sulfuric acid procedure and the cholesterol
oxidase assay. The data on the cellular ceramide content obtained by
the TLC-sulfuric acid procedure were within 5% of those in the
literature, obtained by the diacylglycerol kinase method (53).
Gas-Liquid Chromatography and Mass Spectrometry--
The fatty
acid composition of the total lipids extracted from cell lysates and
from the sphingolipid-enriched sucrose gradient fractions prepared from
cerebellar granule cells at the 8th day in culture was determined by GC
and GC-MS. Samples of the lipid extracts were dissolved in 0.5 ml of
methanol/5 M HCl, 9:1 (v/v), and maintained at 75° for
16 h. Fatty acid methyl esters were extracted three times with 1.5 ml of n-hexane. The hexane solution was dried, and the
residue was dissolved in 50 µl of dichloromethane. Fatty acid methyl
esters were separated on a 25-m SPB-5 capillary column using a
Dani-3865 gas chromatograph, equipped with a program temperature
vaporization injector. Analyses were carried out using a total
injection program, at 5 °C/min, from 180 to 240 °C. Mass spectrometry characterization of the fatty acid methyl esters was
carried out on an hp-5890 GC-MS equipped with a 30-m HP-5 capillary
column. ESI-MS of PC samples, obtained by preparative TLC from cell
lysates and sphingolipid-enriched fraction lipid extracts, was carried
out on a ThermoQuest Finningan LCQdeca mass spectrometer equipped with
an electrospray ion source and a Xcalibur data system. Samples were
dissolved in methanol at a concentration of 2-3 ng/µl and introduced
into the electrospray needle by mechanical infusion through a
microsyringe at a flow rate of 3 µl/min. Ionization was performed
under the following conditions: capillary voltage, 4 keV; sheath gas
flow, 50 arbitrary units; capillary temperature, 260 °C.
Protein Analysis--
Cell lysates and sucrose gradient
fractions obtained after labeling cerebellar granule cells with
[32P]orthophosphate or [35S]methionine were
analyzed to determine protein content and pattern. 35S-Proteins from the sphingolipid-enriched membrane
fractions were analyzed by SDS-PAGE and by two-dimensional
electrophoresis (isoelectric focusing on a pH gradient from 3.5 to 10 in the presence of 9.5 M urea and 1% Nonidet P-40,
followed by 10% acrylamide SDS-PAGE for the second dimension) (30,
54). After separation, proteins were transferred to PVDF membranes, and
radioactive proteins were detected by autoradiography.
The presence of c-Src, Lyn, Fyn, Akt, and PI3K was assessed by
immunoblotting with commercially available specific antibodies, followed by reaction with secondary horseradish peroxidase-conjugated antibody and enhanced chemiluminescence detection (Pierce Supersignal).
Analysis of phosphotyrosine-containing proteins was by
immunoprecipitation (44). Aliquots of the fractions obtained from cells
labeled with [32P]orthophosphate (containing ~15-30
µg of protein) were diluted 10-fold in immunoprecipitation (IP)
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 75 milliunits/ml aprotinin, 1% Triton
X-100). After preclearing for nonspecific binding, 2 µg/ml mouse
anti-phosphotyrosine monoclonal IgG2b or 2 µg/ml normal mouse IgG (as negative control) was added to the supernatants. The
mixtures were placed on a rotary stirrer overnight at 4 °C. Immunoprecipitates were recovered using protein A/G-Sepharose beads,
washed three times with IP buffer, recovered by centrifugation (270 × g, 2 min), suspended in 50 µl of SDS-sample
buffer, heated to 95 °C for 3 min, and centrifuged (1000 × g, 2 min). The radioactivity associated with
immunoprecipitates was determined by liquid scintillation counting. The
total amount of immunoprecipitable radioactivity was calculated for
each fraction and for the cell lysate. Data were expressed for each
fraction as the percentage of total immunoprecipitable radioactivity
present in the amount of cell lysate loaded on gradient. Radioactivity
associated with negative controls never exceeded 5% of radioactivity
found in immunoprecipitates.
Other Analytical Methods--
The radioactivity associated with
cells, with cell fractions, with lipids, and with delipidized pellets
was determined by liquid scintillation counting. Digital
autoradiography of the HPTLC plates and of the PVDF membranes was
performed with a Beta-Imager 2000 instrument (Biospace, Paris) using an
acquisition time of about 24 h. The radioactivity associated with
individual lipids and proteins was determined with the specific
Protein and Lipid Compositions of Rat Cerebellar Granule Cells
Developing in Vitro--
The protein and lipid composition of
cerebellar granule cells at different stages of neuronal development
in vitro are reported, as absolute and relative values, in
Tables I and II. An overall increase in
cell protein and lipid content was observed during the development in
culture. As expected, glycerophospholipids, which represent the bulk
membrane lipids, comprised the highest lipid content of these cells at
all investigated stages of development, 79-82% of total cell lipids.
The glycerophospholipid content significantly increased within the days
in culture. We observed a 2.7-fold increase from the 2nd to the 8th day
in culture, and a further 1.4-fold increase from the 8th to the 17th
day in culture. Within the different glycerophospholipids, PC and PE
were the most abundant at all investigated stages of development (Table
II), being about 50 and 20% of total
glycerophospholipids, respectively. The glycerophospholipid patterns
(Table II) were very similar during development in culture, with two
major exceptions, the plasmalogen species of PE and PC. In
undifferentiated cells, PPC was relatively abundant, representing 11.7% of total glycerophospholipids; its relative amount dramatically dropped to 1-3% at later stages of development. Relative PPE content increased from 3.7% at the 2nd day in culture to 12.6% at the 8th day
in culture (corresponding to a 9-fold increase in the absolute mass
content, unique among all phospholipids) and remained relatively high
at the latest investigated time (6.9%).
Cholesterol was the second major lipid in granule cells at the three
stages of culture (9-18% of total cell lipids, Table I). Its absolute
cellular content increased about 1.7-fold from the 2nd to the 8th day
in culture, and remained almost unchanged later on during development.
Hence, the cholesterol/glycerophospholipids molar ratio markedly
decreased during in vitro development of cerebellar neurons.
Sphingolipids were minor components of undifferentiated cells, being
0.26%, 1.68%, and 0.48% of total cell components in the case of
ceramide, sphingomyelin, and gangliosides, respectively, as shown in
Table I. Their content markedly increased from the 2nd to the 8th day
in culture, as previously reported in the case of gangliosides. This
increase was particularly high (about 11-fold, by mass), from 0.074 nmol/106 cells (0.48% of total cell lipids) at the 2nd day
in culture to 0.79 nmol/106 cells (1.99% of total cell
lipids) at the 8th day in culture, and it was not paralleled by the
increase in glycerophospholipids and cholesterol (Table I). Thus, it
resulted in an increase in the ganglioside/glycerophospholipid and
ganglioside/cholesterol molar ratios. The increases in sphingomyelin
and ceramide (Tables I) were also marked, but less dramatic, from 0.260 nmol/106 cells (1.68% of total cell lipids) and 0.040 nmol/106 cells (0.26% of total cell lipids) at the 2nd day
in culture, to 1.00 nmol/106 cells (2.52% of total cell
lipids) and 0.22 nmol/106 cells (0.55% of total cell
lipids) at the 8th day in culture, respectively. Comparing the
different sphingolipids at the 8th and 17th day in culture, we found
that sphingomyelin content remained unchanged, ganglioside content
increased slightly, and ceramide content increased to a greater extent.
Only in the case of ceramide was the molar ratio of sphingolipid to
glycerophospholipid not decreased.
In agreement with previous reports (56, 57), some remarkable
differences were observed in the relative amounts of the different
gangliosides during the time in culture (Table I).
Palmitic acid was the main fatty acid detected in the total complex
lipid mixture, (40%), followed by oleic acid (34%), and stearic acid
(22%). These data are in good agreement with the fatty acid
composition of PC and PE in differentiated cells, previously reported
in the literature (58).
Lipid Metabolism in Rat Cerebellar Granule Cells Developing in
Vitro--
Fig. 1 shows the patterns of
radioactive lipids extracted from cerebellar granule cells at different
stages of development in culture after HPTLC separation. Data on the
incorporation of radioactivity into different lipid classes are
reported in Table I. The incorporation of radioactivity into
sphingolipids was very similar at all investigated days in culture
(Table I). Keeping in mind the large differences in their endogenous
content, this fact indicates that the sphingolipid turnover is
dramatically different in granule cells at different stages of
development. In particular, it is much more rapid in undifferentiated
cells than later on during development. In fully differentiated cells, the turnover was significantly reduced and remained almost unchanged at
the 17th day in culture. This is particularly evident in the case of
gangliosides: ganglioside-associated radioactivity was almost
identical, whereas their endogenous content increased about 11-fold
from the 2nd to the 8th day in culture.
Among various sphingolipid classes, the incorporation of radioactivity
was not proportional to their mass content even at the same day in
culture. The radioactivity ratio between gangliosides and sphingomyelin
was 12-, 3-, and 4-fold higher than the mass ratio at the 2nd, 8th, and
17th day in culture, respectively, indicating that ganglioside turnover
is always more rapid than sphingomyelin turnover in these cells. This
difference is remarkably high at the early stage of development. The
radioactivity ratio between gangliosides and ceramide was also always
higher than the mass ratio (4-fold at the 2nd and 8th day in culture,
and 1.3-fold at the 17th), indicating a faster turnover for
gangliosides than for ceramide. However, this difference is very small
in cells at the 17th day in culture. Remarkably, these data indicate
that both molar content and turnover of ceramide are significantly increased in cells at a late stage of development. This is in good
agreement with a role of ceramide in cell senescence, as previously
described in the case of fibroblasts (59). Among the different
gangliosides, the distribution of radioactivity in each species closely
reflects the endogenous patterns at all the investigated days in
culture, with the exception of GM3 and GD3 gangliosides in cells at the
2nd day in culture (Table II). The amount of radioactivity incorporated
in these gangliosides represent 13.9 and 30.1% of total ganglioside
radioactivity. The mass content of GM3 was extremely low under these
experimental conditions, and GD3 was 15.1% of total cell gangliosides,
indicating that the turnover of these species is surprisingly rapid. We
are not able to estimate the turnover of neutral glycosphingolipids, because data about their endogenous content in these cells are not
available. No significant differences were observed in the incorporation of radioactivity into GlcCer and LacCer at different days
in culture (about 2 and 5% of total sphingolipid radioactivity, respectively).
The incorporation of radioactivity into
glycerophospholipids, by metabolic labeling with
[32P]orthophosphate, is reported in Table I. The specific
radioactivity of glycerophospholipids was 1.5-fold higher and 3.7-fold
lower at the 2nd and 17th day in culture, respectively, than at the 8th. This indicates that the overall turnover of this lipid class significantly differs in these cells at different stages of development in culture, being slightly higher in undifferentiated cells and markedly lower in cells at the late stage of development, as compared with fully differentiated cells. Remarkably, dramatic differences were
observed among the different glycerophospholipids (Table II). In the
case of PE, PC, and PS (the main cell phospholipids), the incorporation
of radioactivity, expressed as a percentage of the total
[32P]phospholipid radioactivity, was quite constant
during the development in culture, and always lower than the percent
mass content, indicating that their relative turnovers are similar
during this process and relatively slow. On the other hand, the
specific radioactivity of PPC was 3- and 86-fold higher at the 8th day
in culture that at the 2nd and 17th, respectively, indicating very
relevant differences in its turnover. Similar, but less dramatic
differences were observed for phosphatidylinositol. A very rapid
turnover was also observed for phosphatidylinositol plasmalogen and
phosphorylated phosphoinositides, PIP and PIP2. In fact,
these lipids incorporated large amounts of radioactivity, despite their
very low molar content, that made difficult or impossible their
chemical detection. In this case it is difficult to compare their
relative turnover at different stages of development, but it is worth
noting that the incorporation of radioactivity in these lipids is
significantly higher in fully differentiated granule cells than in
undifferentiated or senescent neurons.
Lipid Composition of Sphingolipid-enriched Membrane Fractions from
Rat Cerebellar Granule Cells Developing in Vitro--
Fig.
2 shows the distribution of different
lipids or lipid classes in SEMF prepared from rat cerebellar granule
cells at different stages of development in culture. The fraction of
cellular glycerophospholipids recovered in SEMF was relatively small,
varying from 4.84% of total glycerophospholipids at the 2nd day in
culture to 17.3% at the 17th. Nevertheless, at all the different
stages of development in culture we investigated, glycerophospholipids
were the main lipid component of SEMF, representing 44.2-73.0% of
total SEMF lipids. Because glycerophospholipids account for the bulk of
membrane lipids, these data suggest that the total surface area
occupied by the sphingolipid-enriched domains gradually increases
during development in culture. A relevant (25-40%) amount of cellular cholesterol was associated with SEMF; cholesterol was very abundant in
SEMF in the first stage of culture, about 45% of total SEMF components, then gradually decreased to 15% at 17th day in culture. As
clearly shown, all sphingolipids (namely ceramide, sphingomyelin, gangliosides, and neutral glycosphingolipids) are largely associated with SEMF, regardless from the stage of development (40-70% of the
radioactivity associated with each sphingolipid in the cell homogenate). Thus, these structures are more enriched in sphingolipids than in cholesterol. The incorporation of radioactivity into different lipid classes and the lipid composition of SEMF from cerebellar granule
cells at the 2nd, 8th, and 17th days in culture are reported in Table
III. The data on the cholesterol content
were derived from direct chemical analysis. The data on the
sphingolipid and glycerophospholipid contents were calculated from the
radioactivity distribution in SEMF after labeling cells with
[1-3H]sphingosine or [32P]orthophosphate,
respectively, and from the total lipid composition of these cells, as
reported in Table I. These data clearly indicate that the enrichments
(relative to the homogenates) of SEMF in sphingolipids and cholesterol
with respect to glycerophospholipids linearly decreased with the number
of days in culture. However, the percent lipid composition of SEMF
during development in culture varied in a complex way, as indicated by
the data in Table III. In particular, the relative ganglioside and
sphingomyelin content was higher in fully differentiated cells,
ceramide increased after the initial stage and then remained almost
unchanged, while cholesterol gradually decreased along the days in
culture.
At all the days in culture, the distributions of different gangliosides
within SEMF were very similar to the patterns observed in the
homogenates (Fig. 1 and Table IV), with a
major exception, related to the content of GD1a in SEMF at the 2nd day
in culture. The pattern of radioactive gangliosides observed at the 2nd
day in culture was relatively changeable, compared with those at other stages of development. This variability between different experiments can be easily explained keeping in mind the very rapid variations of
ganglioside content and patterns at this early stage of differentiation (56, 60) and the high variability of the starting biological material
(the age of commercially available newborn rats is determined with a
precision of 12 h, a lapse of time not negligible at this stage of
neonatal development). Despite this variability, GD1a relative content
with respect to total ganglioside at the 2nd day in culture was always
significantly higher in SEMF (an increase of 20-40%) than in the cell
homogenate (Fig. 1).
Fig. 3 shows the distribution of each
glycerophospholipid in SEMF from rat cerebellar granule cells at
different stages of development in culture with respect to the cell
homogenate. Very relevant differences were observed among different
glycerophospholipids and different days in culture. At all the stages
of development, the fraction of PC recovered in SEMF was always much
higher than that of any other glycerophospholipid, thus making PC by
far the most abundant lipid in SEMF (93 and 75% of total SEMF
glycerophospholipids at the 2nd and 17th day in culture, respectively,
Table IV). In the case of other glycerophospholipids, the fraction
associated with SEMF greatly varied among the different days in
culture, thus resulting in very different patterns (Table IV). In
particular, the relative amount of PE in SEMF gradually increased
during differentiation from 2.9% to 26.4%; PPE was particularly high
in cells at the latest stage of development; the amount of
radioactivity associated with phosphorylated phosphoinositides
dramatically increased (especially for phosphatidylinositol
4,5-bisphosphate) during differentiation.
Palmitic acid was the main fatty acid of complex lipids from SEMF.
Palmitic acid was significantly enriched in SEMF respect to the cell
lysate. In fact, its relative content was 1.4-fold higher in SEMF than
in the cell lysate. In parallel, we observed a slight reduction in the
relative amount of unsaturated fatty acids in SEMF. SEMF enrichment in
palmitic acid was mainly due to dipalmitoyl-PC, that represented the
main glycerophospholipid specie in this fraction. PC isolated from the
total cell homogenate and from SEMF was analyzed by ESI-MS according
with previous methods (61). The MS spectrum of PC contained the
molecular ions at m/z 756, 782, 810 corresponding to the
dipalmitoyl-, the 1-palmitoyl-2-oleyl-, and 1-stearoyl-2-oleyl-
species. Fig. 4 clearly shows the large increase in the relative abundance of the molecular ion corresponding to dipalmitoyl-PC, at m/z 756, in SEMF.
Protein Compositions of Sphingolipid-enriched Membrane Fractions
from Rat Cerebellar Granule Cells Developing in Vitro--
Previous
evidence indicated that proteins associated with sphingolipids-enriched
membrane domains are selected and are potentially involved in signal
transduction events (12, 30, 44). Thus, we analyzed the protein
composition of SEMF prepared from cerebellar granule cells at different
stages of development in culture. The protein content of granule cells
increased during culture, as expected, from 26.2 to 85.6 µg/106 cells. To compare these data to those for lipids,
we calculated that at the 2nd, 8th, and 17th day in culture the
approximate molar protein content was 0.44, 1.25 and 1.43 ± 20%
nmol/106 cells, respectively. The SEMF always contained a
very minor portion of cell proteins, but this portion dramatically
increased from the 2nd to the 8th day in culture (0.3 ± 0.1%,
1.6 ± 0.2%, and 2.3 ± 0.3%, of the total cell protein at
the 2nd, 8th, and 17th day in culture, respectively) (Fig.
5, left panel). Fig.
6 shows the two-dimensional
35S-protein patterns of SEMF prepared from granule cells at
different days in culture. The protein pattern in differentiated cells
comprised a wide variety of species, encompassing the whole molecular
mass and isoelectric point range. On the other hand, the protein
patterns of cells at 2nd and 17th DIC were much simpler and
particularly lacking in high molecular mass proteins. A low molecular
mass protein (about 20 kDa) was particularly abundant in SEMF from cells at the latest stage of development.
Fig. 5 (right panel) shows the distribution of
phosphotyrosine-containing proteins in SEMF, as determined by
immunoprecipitation with anti-phosphotyrosine antibody. The portion
of phosphotyrosine-containing proteins associated with SEMF was
relatively high and markedly increased along the development in culture
(7.7%, 22.3%, and 32.1% of total cell phosphotyrosine-containing
proteins at 2nd, 8th, and 17th day in culture, respectively).
Among proteins belonging to the SEMF, three members of the Src family
were identified by immunoblotting with specific antibodies. Fig.
7 shows the distribution patterns of
Src-family tyrosine kinases c-Src, Fyn, and Lyn in SEMF and cell
homogenates from cerebellar granule cells at different stages of
development in culture. From the relative intensities of the
immunoblotting signals, we calculated that the amount of c-Src
associated with SEMF was about 20% of total c-Src present in these
cells, indicating a very high enrichment of c-Src in this fraction. The
amount of c-Src recovered in SEMF was not significantly changed during
the development. Fyn was also highly enriched in SEMF, but the amount of Fyn associated with SEMF increased from 15% of total Fyn at the 2nd
day in culture, to about 45% at the 8th and 17th day in culture. Lyn
was clearly observed in SEMF from cells at the 8th and 17th day in
culture, although the amount of Lyn associated with SEMF was relatively
low (about 5% of the total cell content), but it could not be detected
in SEMF prepared from cells at 2nd day in culture (even loading large
amounts of sample on the gel and prolonging the exposure with the ECL
reagent).
Given the high levels of phosphorylated phosphatidylinositols in SEMF
at some stages of development in culture, and the property of these
cells to remain vital in culture for long time, we analyzed the
distribution patterns of PI3K and of the serine-threonine kinase Akt (a
downstream target of PI3K signaling known to be involved in the control
of neuronal survival), in SEMF and cell homogenates from cerebellar
granule cells at different stages of development in culture. As shown
in Fig. 7, both proteins were expressed in our cells but could not be
detected in the SEMF under our experimental conditions.
Membrane lipids have been reported to change to some extent
during the development, maturation, or aging of the nervous system, or
during the onset of other physiological or pathological changes in
neuronal functions. In our study, the changes in the composition and
turnover of each membrane lipid class and individual lipid during the
development of cultured neurons were related to changes in the
organization of sphingolipid-enriched membrane domains. As a model, we
used primary cultures of rat cerebellar granule cells, which represent
a very homogeneous population of neurons. This highly simplified model
obviously lacks the ability to provide any information about the
interactions between neurons and glia. However, these cells
spontaneously undergo morphological and biochemical changes similar to
those occurring during the different stages of in vivo
neuronal development, thus representing a valuable model for the study
of neuronal differentiation. We performed our experiments at three
different stages of in vitro development: the 2nd day in
culture, corresponding to the initial stage of neuronal differentiation
and neuritogenesis, the 8th day in culture, corresponding to
morphologically and biochemically fully differentiated neurons, and the
17th day in culture, corresponding to a late stage of neuronal
development, followed by the eventual age-induced apoptotic cell death.
The content of all membrane lipid classes (glycerophospholipids,
cholesterol, and sphingolipids) greatly increased during the
progression from undifferentiated to fully differentiated granule
cells, indicating an overall increase in the surface and complexity of
neuronal plasma membrane, corresponding to the extension of a very
elaborate neurite network. This event is also characterized by a great
increase in the cell protein content. This increase is higher in the
case of sphingolipids, particularly gangliosides, than of other lipids,
in agreement with previously reported data (56, 57, 60). Thus, the
surface density of gangliosides in the membrane of fully differentiated neurons is much higher than in undifferentiated cells. The lipid turnover also varied during this phase of development, being higher in
undifferentiated cells both for phospholipids and sphingolipids. The
latter is in agreement with the previous finding that, in these cells,
the activity of 3-ketosphinganine synthase markedly decreases during
differentiation (62). In particular, ganglioside turnover at the 2nd
day in culture was about 11-fold faster than at the 8th day in culture,
whereas in the case of phospholipids this difference was 1.5-fold. The
turnover of gangliosides was always more rapid than sphingomyelin
and ceramide turnover, and this difference was the highest in
undifferentiated cells (12- and 4-fold, for sphingomyelin and ceramide,
respectively). In undifferentiated cells, the turnover of some
ganglioside species, namely GM3 and GD3, was surprisingly high, as
indicated by the high amount of radioactivity associated with these
species compared with their mass content. Moving toward a later stage
of development, resembling that of aging neurons, the density of
gangliosides in the cell membrane showed little or no change, as
indicated by the constant mass ratios between gangliosides and
glycerophospholipids or gangliosides and cholesterol.
The turnover of glycerophospholipids was significantly lower in aging
cerebellar neurons (this fact probably reflects a slower overall
renewal of the plasma membrane), whereas the turnover of gangliosides
was similar to that in fully differentiated cells.
We focused our attention on the organization of sphingolipid-enriched
domains, which are emerging as a compartment within the plasma
membrane, where most cell gangliosides are concentrated and segregated
together with selected proteins and other lipids. The results we
obtained are summarized in Tables III and IV. Our data indicate that
a membrane compartment, characterized by a very high enrichment in
sphingolipids and cholesterol and for this reason named
sphingolipid-enriched domain, always exists in cerebellar granule
neurons, regardless of the stage of development. The bulk structure of
these domains is largely determined by PC, and, within the different PC
species, dipalmitoyl-PC was much more abundant than in the total cell
membrane. Thus, the lipid core of the SEMF results highly enriched in
palmitic acid respect to the cell membrane.
The surface occupied by these structures seems to increase during
development in culture, in a way that is not simply proportional to the
overall cell membrane surface, as indicated by their relative glycerophospholipid content. Moreover, a particularly high ganglioside density (as indicated by the ganglioside/glycerophospholipid mass ratio) within the domains seems to be always required, independent of
the developmental stage. In fact, in undifferentiated neurons, where
the ganglioside content is lower and the neurons are rapidly synthesizing these lipids, we observed in the sphingolipid-enriched domain the highest enrichment in gangliosides with respect to the cell
homogenate, an 8-fold increase in the ganglioside/glycerophospholipid molar ratio. A similar behavior was also observed for sphingomyelin. The SEMF from undifferentiated neurons is also specifically
characterized by the particular enrichment in GD1a ganglioside. The
highest ganglioside/glycerophospholipid mass ratio was measured in
sphingolipid-enriched domains from fully differentiated neurons, which
are also characterized by a relatively high protein content (6-fold
higher than in undifferentiated cells) and a complex protein
composition. Despite the very low protein mass content, SEMF prepared
from neurons at all stages of in vitro development we
investigated contained a significant fraction of cell
phosphotyrosine-containing proteins, that increased during
differentiation. This evidence indicates that only a relatively restricted number of highly selected protein molecules are segregated together in a lipid environment. The data discussed above indicate that
this association is dependent on the developmental status of the neuron
and could be of primary importance in modulating it. In aging neurons,
sphingolipid-enriched domains structure is also characterized by a high
sphingolipid density and protein and phosphotyrosine-containing protein
enrichment. However, in these domains the ganglioside density is
decreased respect to fully differentiated neurons, and the protein
pattern seems to be less complex. It is noteworthy that, in granule
cells at the latest stage of development, the increase in ceramide
levels is higher than those of other sphingolipids. Cerebellar granule
cells after the 17th day in culture undergo massive age-induced death by an apoptotic process. Because ceramide has been widely
implicated as a mediator of cell senescence (59) and programmed cell
death (reviewed in Ref. 7), a ceramide-mediated pathway could be more
relevant in SEMF from aging neurons. Thus, sphingolipid-enriched domains could function as "glycosignaling domains" during
neuronal differentiation and neuritogenesis and as
"ceramide-signaling domains" during neuronal aging and age-induced apoptosis.
If the dynamic association of a specific set of proteins
and lipids within sphingolipid-enriched domains has a functional meaning related to neuronal development, it is very important to
determine which kind of signal is transduced in these units. We focused
our attention on Src family protein-tyrosine kinases. A specific
association of Src-family proteins with gangliosides has been reported
in many cellular types, including nervous system, and has been
implicated in the process of neuronal differentiation. In neuroblastoma
Neuro2a cells, c-Src and its regulatory protein, Csk, are organized in
sphingolipid-enriched membrane domains, and ganglioside-induced
differentiation is mediated by the modulation of c-Src activity within
sphingolipid-enriched membrane domains, followed by mitogen-activated
protein kinase activation (29). In sphingolipid-enriched domains from
fully differentiated rat cerebellar granule cells, Src, Lyn, and Csk
are associated with other proteins and the sphingolipid component of
the domain (30). In particular, the neural cell adhesion molecule TAG-1
is interacting with Lyn, and treatment of cultured cerebellar neurons
with antibodies against GD3 ganglioside or TAG-1 induce Lyn activation,
with consequent tyrosine phosphorylation of an 80-kDa protein (31, 32).
Moreover, in these cells, the artificially induced depletion of
glycosphingolipids at the cell surface is able to disrupt Lyn-mediated
TAG-1 signaling and to interfere with the association of TAG-1 with the
domain (32).
These data strongly support the hypothesis that sphingolipids are
essential components of sphingolipid-enriched membrane domains as
signaling units. The question is, if any naturally occurring changes
in the lipid composition of the domain, such as those we observed
during in vitro development of cerebellar neurons, will lead
to a change in the functional status of signaling proteins in the
domain itself? Our data clearly show that, in cultured cerebellar granule cells at different stages of development, the association of different Src-family proteins with the
sphingolipid-enriched domain follows different patterns. The amount of
c-Src associated with SEMF was very high at all the stages of
development and did not significantly change. Fyn and Lyn association
with SEMF markedly increased during neuronal development, but the
amount of Lyn recovered in the sphingolipid-enriched fraction was much
lower than that of c-Src and Fyn (in the case of undifferentiated
cells, no Lyn was detected in SEMF). Thus, the specific association of
signaling proteins with the domain seems to be dependent on its
structure, as determined by its lipid composition. The translocation of
these proteins from or into the domain, consequent to organizational changes, could be a mechanism of regulation. In Neuro2a neuroblastoma cells, the activation of c-Src in the sphingolipid-enriched domain, that mediates ganglioside-induced neuritogenesis, is accompanied by the
removal of Csk from the domain (29). Because Csk is an inhibitory
regulatory kinase of c-Src, this is a good example of regulation
mediated by an alteration of sphingolipid-enriched domain structure.
Thus, it will be very interesting to investigate whether the activity
of these proteins within sphingolipid-enriched domains actually changes
during in vitro development and whether the transient
association with other protein molecules could be responsible for this change.
More specific differences in the levels, turnover, and
organization of single lipid species were observed. Some of them are intriguing, even if still difficult to interpret. For example, a single
ganglioside, GD1a, is significantly more enriched than others in
sphingolipid-enriched domains from undifferentiated cells. This could
indicate a specific, yet unknown role for GD1a. PPE is particularly
enriched in sphingolipid-enriched domains from aging neurons, and
several lines of evidence indicate that the receptor-mediated breakdown
of plasmalogens is a relevant event in neural membranes during
neurodegeneration (8). The turnover of phosphorylated phosphoinositides
is particularly intense in sphingolipid-enriched domains from fully
differentiated neurons. Thus, lipid-mediated signaling pathways other
than sphingolipid signaling could play a role within these domains.
This will not be surprising, because our data clearly indicate that
non-sphingoid lipids also undergo a selection process leading to the
segregation within domains. Nevertheless, neither the phosphoinositide
3-kinase nor the serine-threonine kinase Akt, which is known to be one of the phosphorylated phosphoinositide signaling downstream targets, were detected in the sphingolipid-enriched domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid-ergic, inhibitory interneurons. Rat
cerebellar granule cells in culture immediately start to differentiate
and spontaneously undergo a developmental pattern that resembles that of cerebellar neurons in vivo, but in a condensed time
scale, reaching a mature state after 8 days. The experiments were
performed at three different stages of neuronal development in culture. The 2nd day in culture corresponded to the initial stage of neuronal differentiation and neuritogenesis. At this stage, the cells already emit short neurite-like processes (38). The 8th day in culture corresponded to morphologically and biochemically fully differentiated neurons; the cells are mostly grouped in large aggregates, connected by
a complex net of fasciculated fibers. The progressive formation of
axo-axonic synapses was observed. From the biochemical point of view,
granule cells at this stage of in vitro development are characterized by the expression of voltage-dependent sodium
channels, neurotransmitter receptors, neuronal surface
sialoglycoproteins. Moreover, they acquire the ability to synthesize
and release glutamate under depolarizing conditions (36, 39). The 17th
day in culture corresponded to a late stage of neuronal development,
characterized by the onset of age-induced apoptotic death of cerebellar
granule cells (40). Thus, the primary cultures are a valuable model for
the study of neuronal development in vitro. Typical protein content at these times was 240, 700, and 770 µg of protein/dish.
8 M
[1-3H]sphingosine (5 ml/dish) in cell-conditioned medium
for a 2-h pulse followed by a 48-h chase. Under these conditions, free
radioactive sphingosine was hardly detectable in the cells, and all
sphingolipids and phosphatidylethanolamine (obtained by recycling of
radioactive ethanolamine formed in the catabolism of
[1-3H]sphingosine) were metabolically radiolabeled (30,
44). Cells at the 1st, 7th, and 16th day in culture were preincubated
in methionine-free medium for 2 h and subsequently incubated in
the presence of 25 µCi/ml L-[35S]methionine
(5 ml/dish) for 20 h, to achieve steady-state radiolabeling of
proteins (44-46). Cells at the 2nd, 8th, and 17th day in culture were
incubated in the presence of 50 µCi/ml carrier-free
[32P]orthophosphate (5 ml/dish) in phosphate-free culture
medium for 4 h (44, 47). These experimental conditions allowed
introduction of radioactivity into phosphoproteins and
glycerophospholipids, these latter including the very minor ones, such
as the phosphorylated phosphoinositides (47).
-Vision software provided by Biospace. Autoradiography of
32P- and 35S-labeled proteins was carried out
using Kodak Biomax MR and MS films. Protein content was determined
according to Lowry (55) using bovine serum albumin as reference standard.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Protein and lipid composition and radioactivity incorporation in rat
cerebellar granule cells during development in culture
Ganglioside and glycerophospholipid species composition and
radioactivity incorporation in rat cerebellar granule cells during
development in culture
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Fig. 1.
Patterns of radioactive lipids in homogenates
and sphingolipid-enriched membrane fractions from rat cerebellar
granule cells at different stages of development in culture after
feeding [1-3H]sphingosine. Lipids from the
homogenates (Hom) and from the sphingolipid-enriched
membrane fractions (SEMF) prepared from rat cerebellar
granule cells at the 2nd (A), 8th (B), and 17th
(C) day in culture were extracted as described under
"Experimental Procedures" and separated by HPTLC in the solvent
system chloroform/methanol/0.2% CaCl2 50:42:11 (v/v).
Radioactive lipids were detected by digital autoradiography (250 dpm
applied on a 4-mm line. Time of acquisition: 24 h). Pattern is
representative of that obtained in three different experiments.
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Fig. 2.
Lipid distribution in the
sphingolipid-enriched membrane fractions prepared from rat cerebellar
granule cells at different stages of development in culture.
Relative amounts of ceramide (Cer), sphingomyelin
(SM), gangliosides (G), neutral
glycosphingolipids (NGSL), glycerophospholipids
(GPL), and cholesterol (Chol) recovered in the
sphingolipid-enriched membrane fractions prepared from rat cerebellar
granule cells at the 2nd (light gray), 8th
(black), and 17th (dark gray) day in culture.
Data relating to sphingolipids and glycerophospholipids were obtained
from the distribution of lipid-associated radioactivity after labeling
with [1-3H]sphingosine or
[32P]orthophosphate, respectively. Endogenous cholesterol
content was directly determined as described under "Experimental
Procedures." Data are expressed as percentages of the total amount of
each lipid present in the homogenates, and are the means of three
different experiments ± S.D.
Protein and lipid composition of the sphingolipid-enriched membrane
fraction from rat cerebellar granule cells during development in
culture
Ganglioside and glycerophospholipid species composition of the
sphingolipid-enriched membrane fraction from rat cerebellar granule
cells during development in culture
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Fig. 3.
Phospholipid distribution in the
sphingolipid-enriched membrane fractions prepared from rat cerebellar
granule cells at different stages of development in culture.
Relative amounts of phosphatidylethanolamine (PE),
phosphatidylethanolamine plasmalogen (PPE),
phosphatidylcholine (PC), phosphatidylcholine plasmalogen
(PPC), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylinositol plasmalogen
(PPI), phosphatidylinositol-4-phosphate (PIP),
phosphatidylinositol 4,5-diphosphate (PIP2)
recovered in the sphingolipid-enriched membrane fractions prepared from
rat cerebellar granule cells at the 2nd (light gray), 8th
(black), and 17th (dark gray) day in culture.
Data were obtained from the distribution of lipid-associated
radioactivity after labeling with [32P]orthophosphate.
Lipids from the homogenates and from the sphingolipid-enriched membrane
fractions were extracted and separated by mono- and bidimensional HPTLC
as described under "Experimental Procedures." Radioactive lipids
were detected by autoradiography (typically 2000 cpm applied on a 3-mm
line. Time of exposure: 48 h). The radioactivity associated with
individual lipids was determined by densitometry. Data are expressed as
percentages of the total amount of each lipid present in the
homogenates and are the means of three different experiments ± S.D.
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Fig. 4.
Positive-ion ESI mass spectra of PC from
cerebellar granule cells at the 8th day in culture. PC was
purified from the total lipid extracts obtained from the homogenate and
the sphingolipid-enriched membrane fractions from cells at the 8th day
in culture as described under "Experimental Procedures."
A, mass spectrum of PC from cell homogenate; B,
mass spectrum of PC from the sphingolipid-enriched membrane fraction.
Data are expressed as percent relative abundances. The three main
species dipalmitoyl-PC, 1-palmitoyl-2-oleyl-PC, and
1-stearoyl-2-oleyl-PC with molecular ions [M+Na+] at
m/z 756, 782, and 810, respectively, were recognized. Each
selected ion was characterized by MS2 and MS3
by loss of quaternary amino and phosphoethyl groups,
respectively.
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Fig. 5.
Protein and
phosphotyrosine-containing protein distributions in the
sphingolipid-enriched membrane fractions prepared from rat cerebellar
granule cells at different stages of development in culture.
Relative amounts of radioactivity associated with proteins (left
panel) and with phosphotyrosine-containing proteins (right
panel) in the sphingolipid-enriched membrane fractions prepared
from rat cerebellar granule cells at the 2nd (light gray),
8th (black), and 17th (dark gray) day in culture
after labeling with [35S]methionine or
[32P]orthophosphate, respectively. Radioactivity was
determined by liquid scintillation counting. Data are the means of
three different experiments ± S.D. The amounts of radioactivity
associated with proteins are expressed as percentages of the total
amount present in the homogenates. Radioactivity associated with
phosphotyrosine-containing proteins was determined after
immunoprecipitation with anti-phosphotyrosine monoclonal antibody as
described under "Experimental Procedures," and data are
expressed as the percentage of total immunoprecipitable radioactivity
present in the homogenate.
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Fig. 6.
Two-dimensional protein patterns in
sphingolipid-enriched membrane fractions from rat cerebellar granule
cells at different stages of development in culture, after labeling
with [35S]methionine. Similar amount of proteins
from sphingolipid-enriched membrane fractions prepared from rat
cerebellar granule cells at the 2nd (A), 8th (B),
and 17th (C) day in culture after steady-state metabolic
labeling with [35S]methionine were analyzed by
two-dimensional electrophoresis, followed by autoradiography (about
5000 cpm; time of exposure: 15 days) as described under "Experimental
Procedures." Patterns are representative of those obtained in three
different experiments.
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Fig. 7.
Signal transduction proteins in the
sphingolipid-enriched membrane fractions and homogenates prepared from
rat cerebellar granule cells at different stages of development in
culture. The same percentage of the total from the
sphingolipid-enriched membrane fractions (SEMF) and from the
homogenates (Hom) prepared from rat cerebellar granule cells
at the 2nd (A), 8th (B), and 17th (C)
day in culture were analyzed by SDS-PAGE followed by detection by
Western blotting using specific anti-c-Src, anti-Lyn, anti-Fyn,
anti-PI3K, and anti-Akt antibodies, as indicated at the left of
each panel. Patterns are representative of those obtained in three
different experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This research was supported by the Cofinanziamento Ministero dell' Università e delle Ricerca Scientifica e Tecnologica Progetti di Interesse Nazionale (MURST PRIN) 1997 (to S. S. and G. T.), Cofinanziamento MURST PRIN 1998 (to V. C.), and by Consiglio Nazionale delle Ricerche (Target project Biotechnology to G. T. and S. S.), Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Permanent address: Dept. of Chemistry, Saint Lawrence University,
Canton, NY 13617.
§ To whom correspondence should be addressed: Dipartimento di Chimica e Biochimica Medica-LITA-Segrate, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy. Tel.: 39-02-2642-3204; Fax: 39-02-2642-3209; E-mail: Sandro.Sonnino@unimi.it.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M010666200
2
The ganglioside and glycosphingolipid
nomenclature is in accordance with Svennerholm (63) and the IUPAC-IUBMB
recommendations (64). The abbreviations used are: GlcCer,
-Glc-(1-1)-Cer; LacCer,
-Gal-(1-4)-
-Glc-(1-1)-Cer; GM3,
-Neu5Ac-(2-3)-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GM1,
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GD3,
-Neu5Ac-(2-8)-
-Neu5Ac-(2-3)-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GD1a,
-Neu5Ac-(2-3)-
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GD1b,
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5Ac-(2-8)-
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer; O-Ac-GT1b
-Neu5Ac-(2-3)-
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5,9Ac2-(2-8)-
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GT1b,
-Neu5Ac-(2-3)-
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5Ac-(2-8)-
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer; GQ1b,
-Neu5Ac-(2-8)-
-Neu5Ac-(2-3)-
-Gal-(1-3)-
-GalNAc-(1-4)-[
-Neu5Ac-(2-8)-
-Neu5Ac-(2-3)]-
-Gal-(1-4)-
-Glc-(1-1)-Cer.
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ABBREVIATIONS |
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The abbreviations used are: GPI, glycosylphosphatidylinositol; SEMF, sphingolipid-enriched membrane fraction; Cer, ceramide, N-acyl-sphingosine; [1-3H]sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-[1-3H]octadecene; PC, phosphatidylcholine; LPC, lyso-phosphatidylcholine; PPC, phosphatidylcholine plasmalogen; PS, phosphatidylserine; PE, phosphatidylethanolamine; PPE, phosphatidylethanolamine plasmalogen; PI3K, phosphoinositide 3-kinase; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; HPTLC, high performance thin-layer chromatography; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; GC, gas chromatography; MS, mass spectrometry; ESI, electrospray ionization; DIC, Day(s) in culture.
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