Am J Physiol Lung Cell Mol Physiol 287: L743–L751, 2004.
First published May 28, 2004; 10.1152/ajplung.00146.2004.
Surfactant lipid synthesis and lamellar body formation in glycogen-laden
type II cells
Ross Ridsdale and Martin Post
Canadian Institutes of Health Research Group in Lung Development, Hospital for Sick Children Research
Institute, and Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Submitted 22 April 2004; accepted in final form 26 May 2004
CTP:phosphocholine cytidylyltransferase; pulmonary surfactant; immunogold; fatty acid synthase
SHORTLY BEFORE BIRTH, alveolar type II cells must increase their
surfactant lipid and protein synthesis to generate sufficient
pulmonary surfactant. This material reduces the surface tension
at the air/liquid interface and ample amounts are required to
prepare the lung for extrauterine life (30). Surfactant production appears to be a carefully timed event because a preterm
infant can experience respiratory distress and atelectasis as a
direct result of pulmonary surfactant insufficiency.
Pulmonary surfactant is composed of a mixture of lipids and
proteins. The most abundant lipid species is phosphatidylcholine (PC) with dipalmitoyl-PC being the surface-active reagent
(17). Whereas alveolar type II cells in the postpartum lung
need to synthesize lipids and proteins for pulmonary surfactant
maintenance, the fetal type II cells need to generate enough
material to establish a functioning air-liquid barrier immediately at birth (10, 29). It is evident that the surge of production
of surfactant at late fetal gestation is a daunting metabolic task
for the type II cells. Morphologically, large amounts of glycogen accumulate in the distal undifferentiated epithelium before
Address for reprint requests and other correspondence: M. Post, Lung
Biology Program, Hospital for Sick Children, 555 University Ave., Toronto,
Ontario, Canada M5G 1X8 (E-mail: [email protected]).
http://www.ajplung.org
surfactant synthesis, and these glycogen stores are depleted as
surfactant is synthesized (6, 32). Biochemically, temporal relationships between glycogen and phospholipids (5, 7) have
suggested that glycogen is used as a carbon source to generate
the glycerol backbone and acyl chains of the surfactant lipids,
but a direct precursor-product relationship has not been demonstrated. Furthermore, it has been suggested that glycogen
acts as an energy source for the rapidly maturing type II cells
(25). Thus pretype II cells build up large stores of the glucose
substrate glycogen to support the rapid metabolic process of
increased surfactant lipid synthesis close at term.
One, usually unappreciated, consequence of these large
glycogen stores is the profound effect they have on fetal type
II cell morphology (21). The volume of carbohydrate in a
preterm type II cell causes swelling and reduces the cytoplasm.
The large stores of glycogen are not membrane bound and are
devoid of organelles, such as endoplasmic reticulum (ER),
Golgi apparatus, or mitochondria. The exclusion of these
organelles indicates that this glycogen is a distinct microenvironment from the surrounding cytoplasmic space. It is unclear
how type II cells in late gestation are able to synthesize the
necessary amount of lipid when the commonly accepted subcellular sites for synthesis, ER and Golgi, are marginalized to
small cytoplasmic pockets that are separated by glycogen.
To improve our understanding of surfactant production during late fetal gestation, we investigated the cellular localization
of two key enzymes for PC synthesis, fatty acid synthase
(FAS), and CTP:phosphocholine cytidylyltransferase (CCT␣), in type II cells of fetal mice (19, 53, 54). Their localization
was compared with that of the cellular surfactant storage
organelles, the lamellar bodies (LBs), and surfactant protein
(SP)-A, -B, and -C. We observed that the glycogen stores are
inhabited by lipid-synthesizing enzymes, surfactant proteins,
and surfactant storage organelles. Energy-filtering electron
microscopy (EM) revealed the presence of phosphorous-rich
domains in the glycogen and the binding of CCT-␣ to these
domains. Our observations suggest that the glycogen is an
appropriate environment for surfactant lipid synthesis and LB
formation/maturation and substantiate that the glycogen functions as a substrate for surfactant phospholipids.
MATERIALS AND METHODS
Materials. C57BL/6 mice were obtained from Charles River (St.
Constant, Quebec, Canada). All mouse protocols were in accordance
with Canadian Council on Animal Care guidelines and were approved
by the Animal Care and Use Committee of the Hospital for Sick
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.
1040-0605/04 $5.00 Copyright © 2004 the American Physiological Society
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Ridsdale, Ross, and Martin Post. Surfactant lipid synthesis and
lamellar body formation in glycogen-laden type II cells. Am J Physiol
Lung Cell Mol Physiol 287: L743–L751, 2004. First published May
28, 2004; 10.1152/ajplung.00146.2004.—Pulmonary surfactant is a
lipoprotein complex that functions to reduce surface tension at the air
liquid interface in the alveolus of the mature lung. In late gestation
glycogen-laden type II cells shift their metabolic program toward the
synthesis of surfactant, of which phosphatidylcholine (PC) is by far
the most abundant lipid. To investigate the cellular site of surfactant
PC synthesis in these cells we determined the subcellular localization
of two key enzymes for PC biosynthesis, fatty acid synthase (FAS)
and CTP:phosphocholine cytidylyltransferase-␣ (CCT-␣), and compared their localization with that of surfactant storage organelles, the
lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse
lung. Ultrastructural analysis showed that immature and mature LBs
were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron
microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-␣. Energy-filtering EM demonstrated that CCT-␣ bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized
predominantly to the glycogen stores. Collectively, these data suggest
that the glycogen stores in fetal type II cells are a cellular site for
surfactant PC synthesis and LB formation/maturation consistent with
the idea that the glycogen is a unique substrate for surfactant lipids.
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SURFACTANT FORMATION IN FETAL LUNG CELLS
resulting blocks were cut using a diamond knife on a Reichert Ultracut
microtome to gold thickness and placed onto 400-mesh copper grids.
The samples were stained 10 min in 3% (wt/vol) uranyl acetate in
double-distilled water, 5 min in 1% (wt/vol) lead citrate, followed by
several washes with double-distilled water to remove excess stain.
The samples were examined on a transmission electron microscope
(model 430, Philips).
Freeze substitution. The 1-mm tissue pieces were briefly coated
with 20% (wt/vol) sucrose in PBS for cryoprotection and rapidly
frozen in liquid nitrogen. The tissues were permitted to dehydrate
slowly in 3% (wt/vol) uranyl acetate in 100% methanol at ⫺70°C over
5 days. The methanol was switched to 1:1 lowicryl HM20:100%
methanol by three incubations of 3 h at ⫺20°C, followed by three
changes of 100% lowicryl HM20 (3 h per change). The tissue was
then placed in HM20 lowcryl (Marivac) containing beam capsules and
exposed to UV light for 2 days at ⫺20°C to polymerize the lowicryl.
The polymerized blocks were removed from the capsules and allowed
to dry at room temperature for 1 day. Ultrathin sections of the tissues
were cut using a diamond knife on a Reichert Ultracut microtome to
light gold thickness and floated onto formvar coated 300-mesh nickel
grids for immunogold labeling.
Immunogold labeling. Sections on the nickel grids were washed
three times in double-distilled filtered water (10 min per wash) and
then blocked with 5% (wt/vol) glycine in PBS (10 min), followed by
three washes in 5% (wt/vol) powdered milk in PBS (10 min each).
Primary antibodies were added to the samples diluted in 1% (wt/vol)
BSA in PBS. The final concentrations of the primary antibodies were
1:200 for anti-FAS, 1:100 for anti-CCT-␣, 1:100 for anti-SP-A, 1:100
for anti-SP-B, and 1:200 for anti-pro-SP-C. After 2-h incubation at
room temperature, samples were washed four times in PBS (10 min
per wash) and then incubated for 2 h with 1:300 secondary gold (5 or
10 nm)-conjugated goat anti-rabbit IgG (Nanoprobes, Yaphank, NY)
Fig. 1. Specificity of primary and secondary
antibodies. A: Western blots of embryonic
day 18 (E18) whole lung tissue homogenate
using primary rabbit antibodies against either fatty acid synthase (FAS), CTP:phosphocholine cytidyltransferase-␣ (CCT-␣),
surfactant protein-A and -B (SP-A and -B),
or pro-SP-C. B and C: immunogold labeling
of E18 fetal mouse type II cell with goldlabeled secondary anti-rabbit IgG using no
primary antibody (C) or with preimmune
rabbit serum (B). Scale bar ⫽ 500 nm.
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Children. Female mice were bred with male sires and checked for
plugs. At embryonic day 18 (E18), fetuses were collected from the
mothers who were first euthanized by cervical dislocation. Fetal lungs
were extracted and minced into ⬃1 mm pieces. Tissue was prepared
for EM by either routine fixation for basic histology or freeze
substitution for immunogold examination. A rabbit polyclonal antibody against the NH2-terminal domain of CCT-␣ was raised as
previously described (31, 51). Rabbit polyclonal FAS antibody was a
generous gift from S. Rooney (Yale University, New Haven, CT).
Rabbit polyclonal antibodies for SP-A and SP-B were purchased from
Chemicon (Temecula, CA). Rabbit polyclonal pro-SP-C antibodies
were generously provided by M. Beers (University of Pennsylvania,
Philadelphia, PA). The specificity of the antibodies was determined by
Western blot analysis of E18 whole lung homogenates. Aliquots of
homogenate (80 ␮g of total protein) were separated by SDS-PAGE,
transferred to membranes, immunoblotted with primary antibody,
followed by detection with a horseradish peroxidase-linked secondary
antibody and an enhanced chemiluminescence system (Amersham,
Piscataway, NJ). The antibodies recognized single or multiple bands
with reported molecular ratio positions for FAS (26, 50), CCT-␣ (31),
SP-A (22, 23), SP-B (9, 11), and pro-SP-C (3) (Fig. 1A).
Routine histology. Tissues were fixed in 4% (vol/vol) paraformaldehyde (Sigma; St. Louis, MO), 0.5% (wt/vol) glutaraldehyde
(Sigma) in PBS for 2 h. Tissues were then rinsed three times with
PBS, exposed to 1% (wt/vol) osmium tetroxide (Marivac) for 1 h,
followed by three more washes with PBS. The tissues were then
dehydrated through an ascending alcohol series and then incubated
three consecutive times in propylene oxide (Merivac) for 30 min. The
propylene oxide was then replaced with a 1:1 mixture of propylene
oxide and Epon (Marivac) for 2 h and then finally three times with
100% Epon. The Epon-infiltrated tissues were placed in molds and the
Epon was polymerized at 70°C overnight. Ultrathin sections of the
SURFACTANT FORMATION IN FETAL LUNG CELLS
RESULTS AND DISCUSSION
Fetal lung epithelial type II cells are ultrastructurally distinct
from mature adult type II cells. In particular, fetal type II cells
contain large glycogen stores, which are absent in fully differentiated type II cells (Fig. 2, A and C). In these glycogen-laden
cells, the glycogen is concentrated at the apical side of the cell
(Fig. 2, C–E). Intracellular organelles such as ER, Golgi
apparatus, and mitochondria were not visible within the glycogen stores. The ER is distributed throughout the available
cytoplasmic space. Mitochondria appear to be mainly, but not
exclusively, concentrated to the basolateral regions (Fig. 2E).
Thus the abundant glycogen severely marginalizes the cytoplasm of the type II cells at late fetal gestation. At the same
time, these glycogen-laden cells must produce the surfaceactive material required for extrauterine air breathing. To better
characterize the surfactant production in these cells, we chose
to investigate the subcellular localization of FAS and CCT-␣,
two key enzymes for surfactant PC synthesis (19, 53) and
relate their localization to LBs and related structures as well as
surfactant proteins.
Initially, we examined the subcellular localization of LBs
and multivesicular bodies (MVB). Although the glycogen
stores are devoid of ER, Golgi, and mitochondria, LBs of
varying sizes are frequently observed in them (Fig. 2, D and E).
Incidental examples of LBs within the glycogen-rich zones
have often been reported without major discussion (14, 16, 30,
34, 38, 47). Clearly, the glycogen stores do not represent a
barrier to LBs or at least some types of lipid-rich structures.
The appearance of small, apparently immature LBs (Fig. 3, B
and C) suggest that these structures could develop in the
glycogen stores.
Interestingly, the glycogen-laden epithelium was devoid of
multivesicular bodies (Fig. 2E). Such structures are involved in
delivering recycled surfactant proteins and lipids to LBs (45,
46). Their involvement in transporting newly synthesized surfactant lipids to LBs is less clear (43). The apparent absence of
MVB within the glycogen suggests that either MVBs are not
detectable when distributed within the glycogen or these structures are not essential for LB formation at this stage of
development. It is possible that the relatively low contrast of
MVBs in the cytoplasm of type II cells makes them irresolvable in the glycogen. It is unknown what role MVBs play in
newly synthesized lipid incorporation into LBs. Radioautographic studies (8) have shown that PC transport to LBs was
AJP-Lung Cell Mol Physiol • VOL
independent of MVBs, suggesting that there is a PC transport
pathway independent of MVBs (44). Furthermore, MVB do
not osmicate as well as LBs, resulting in a low-contrast,
electron-lucent appearance. This differing response to osmium
is indicative of a higher saturated lipid content in the MVBs.
Although there is no direct evidence of recycling, the osmification difference suggests a different lipid composition between LBs and MVBs. This finding substantiates the idea that
some LB lipids are derived via an MVB-independent transport
pathway. In addition, the infrequency of MVBs in fetal epithelial cells suggests that these organelles may not be essential
to LB formation.
We then investigated whether the glycogen stores are a
potential assembly site for LB formation. With so little cytoplasmic space available in some of these cells, it is unlikely
LBs could form anywhere else. First, we decided to examine
the subcellular localization of CCT-␣ to identify glycogen as a
potential site for PC synthesis. CCT-␣ catalyzes the rate
limiting step in the CDP choline or Kennedy pathway, the
major pathway for de novo PC synthesis in most mammalian
cells (20). Previously, we have shown that increases in surfactant PC production by fetal type II cells are associated with
increases in CCT-␣ protein content and activity (19, 53). We
have also reported that Flag-tagged CCT-␣ distributes to the
glycogen-filled compartments of distal epithelium in mice
overexpressing Flag-tagged CCT-␣ (31). Herein, we show that
endogenous CCT-␣ also distributes to the glycogen stores (Fig.
3). CCT-␣ is found bound to ER in the cytoplasm (Fig. 3A,
black arrows) and to the limiting membrane of LBs in the
glycogen pools (Fig. 3A). Occasionally, CCT-␣ was associated
with extracellular LBs (Fig. 3A, inset). Although CCT-␣ is not
likely secreted for any functional purpose, it does suggest that
the lipid association is strong. The observation of CCT-␣ being
bound to lipid-rich LBs suggests that CCT-␣ may be active
within the glycogen pool. Membrane association of CCT-␣ has
been shown to increase catalysis by decreasing the Km for CTP
(52) and increasing the catalytic constant (15). Traditionally,
the cellular site for CCT-␣ activity has been thought to be the
ER; however, several reports now indicate that CCT-␣ may by
active within the nucleus (13, 27, 41, 42). Similar to previous
studies (31, 39), we observed no CCT-␣ signal in the nucleus
(Fig. 3D), corroborating that in alveolar type II cells CCT-␣ is
entirely extranuclear. Thus our data suggest that CCT-␣ in
glycogen-laden type II cells may be active in another subcellular compartment other than the ER. CCT-␣ also localized to
some of the smaller structures present within the glycogen
(Fig. 3, B and C, and 4B). Many of these structures display
multiple lipid layers that could be immature LBs (Fig. 3, B and
C, black arrowheads). To confirm that these structures contained phospholipids, we performed energy-filtering transmission EM (EFTEM). With this technology, it is possible to
selectively image a sample for regions containing particular
atoms. In our case, we examined the membrane/CCT-␣ boundaries by exploiting the high phosphorus content found in
membrane phospholipids versus the high nitrogen content
found in protein. As shown in Fig. 3, some small multileaflet
structures were labeled by immunogold for CCT-␣. These
structures displayed a strong phosphorus signal (Fig. 4A),
suggesting that the structures have a high phospholipid content.
This signal was similar to that seen with larger LBs and
strongly suggests that small and large LBs are found within the
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diluted in 1% (wt/vol) BSA in PBS. Sections were washed four times
in PBS and then two times in double-distilled water, 10 min per wash.
The samples were either examined without stain or stained 10 min in
3% (wt/vol) uranyl acetate in double distilled water, 5 min in 1%
(wt/vol) lead citrate, followed by several washes with double-distilled
water to remove excess stain. Samples were examined on a Philips
430 electron microscope. Routine negative controls included omission
of primary antibodies and samples treated with preimmune serum. In
all cases, no applicable gold labeling was observed in these controls
(Fig. 1, B and C).
Elemental spectral analysis. Routine or immunogold labeled sections were analyzed using a Philips Tecnai 20 transmission electron
microscope fitted with a Gatan electron imaging spectrometer (2). Net
phosphorus and nitrogen maps were produced from dividing preedge
elemental image (120 eV for phosphorus and 385 eV for nitrogen) and
postedge elemental image (155 eV from phosphorus and 415 eV for
nitrogen).
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SURFACTANT FORMATION IN FETAL LUNG CELLS
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Fig. 2. Lamellar bodies (LBs) and glycogen in transmission electron microscopy (TEM) histology. a: type
II cell with LBs from adult mouse lung. b: higher
magnification of LBs of adult type II cell. Some multivesicular bodies (MVB) are visible at the top left.
c: E18 murine type II cells containing differing amounts
of glycogen. d: another E18 murine type II cell with less
glycogen than the previous sample. Note the LB inside
the glycogen. e: glycogen-filled fetal type II cells processed for immunogold. Note the LBs in the glycogen.
Endoplasmic reticulum (ER) (indicated by arrow), mitochondria (Mit), and the nucleus (Nuc) are separated
from the glycogen (*), and the available cytoplasm is
marginalized. Scale bar ⫽ 500 nm.
glycogen stores (Fig. 4, D and E). Energy filtering of glycogenladen type II cells resulted in some other unexpected findings
(Fig. 4, C–E). The outer boundary of the glycogen, which
appears indistinct from the overall glycogen pool, exhibits a
high concentration of phosphorus and a weaker nitrogen signal
(Fig. 4, D and E, white arrows). This suggests that there are
cellular structures extending into the glycogen that are not
resolvable by regular TEM. Furthermore, there is an increase in
AJP-Lung Cell Mol Physiol • VOL
nitrogen on a confined region of the LB that is facing the basal
lateral side of the cell (Fig. 4, D and E, white arrows). This
protein-rich region has not been previously described and
could play a role in transport and/or membrane fusion.
The CCT-␣ immunogold findings do not resolve the issue
whether pulmonary surfactant lipids and LBs are solely manufactured within the glycogen or whether small lipid membrane structures are generated outside the glycogen, which then
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SURFACTANT FORMATION IN FETAL LUNG CELLS
move into the glycogen and contribute to LB formation. Some
ER-bound CCT-␣ was observed in these samples (Fig. 3A,
black arrows), consistent with the idea that some PC is being
synthesized outside the glycogen. We speculate that ER-bound
CCT-␣ aid, the production of PC for membrane biogenesis.
However, the presence of membrane-bound CCT-␣ in the
glycogen is a strong indicator that the rate-limiting enzyme for
surfactant PC synthesis (36) is also active within the glycogen
storage pools. Choline kinase (CK) catalyzes the first reaction
in the CDP-choline pathway yielding phosphocholine from
choline and ATP. CK is a cytosolic activity in most cells (49).
Where CK localizes in fetal type II cells remains to be
determined, but its product, phosphocholine, should diffuse
freely into the glycogen compartments together with CTP. The
availability of both substrates in the glycogen supports the idea
that CCT-␣ within the glycogen is functionally active. The
product (CDPcholine) of the CCT-␣ catalyzed reaction is
hydrophilic and should distribute freely in and out of the
glycogen. In the final step of the Kennedy pathway CDPchoAJP-Lung Cell Mol Physiol • VOL
line condenses with diacyglycerol to form PC, a reaction
catalyzed by cholinephosphotransferase (CPT). This enzyme is
a transmembrane protein that has been localized to the ER,
Golgi, and nuclear membranes (18). In a previous report (1), no
CPT activity was found to be associated with LBs, suggesting
that LBs were not involved in the final step of PC formation.
Unfortunately, the authors did not analyze the activity of
CCT-␣. Whether the CPT findings obtained with LBs isolated
from adult mouse lung do reflect the immature and growing
LBs of the fetal type II cells is unknown. It is evident that an
ultrastructural location of choline phosphotransferase in glycogen-laden type II cells would provide a more conclusive insight
into the major sites for PC synthesis in these cells. However, to
our knowledge, no reliable antibody has yet been produced for
this protein.
The temporal relationship between glycogen and phospholipid during lung development (5, 7) suggests that glycogen
serves as a carbon source to generate the glycerol backbone
and acyl chains of the surfactant glycerolipids. De novo fatty
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Fig. 3. CCT-␣ is associated LBs within the
glycogen. a: E18 type II cell immunogold
labeled for CCT-␣. CCT-␣ is distributed
throughout the glycogen and is bound to
LBs. Some CCT-␣ is found in the cytoplasm
bound to ER (arrows). A secreted LB is also
labeled by CCT-␣ immunogold (arrowhead
and inset). b and c: small lamellar structures
inside the glycogen are also decorated with
several immunogold particles (arrowheads).
d: typical view of a E18 type II cell labeled
with immunogold for CCT-␣ with a clear
view of the nucleus. Two gold particles are
visible in the cytosol (arrowheads), but no
gold particles are observed in the nucleus.
Scale bar ⫽ 100 nm.
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SURFACTANT FORMATION IN FETAL LUNG CELLS
acid biosynthesis depends on the activity of FAS, the enzyme
that catalyzes the final steps in fatty acid synthesis (35). During
lung development, FAS exhibits a similar activity profile as
CCT-␣ (4, 24). To find out whether the glycogen deposits in
the maturing type II cells are a major site of de novo fatty acid
biosynthesis, we examined the subcellular localization of FAS.
Immunogold EM identified the glycogen as the primary and
nearly exclusive site for FAS (Fig. 5A). Although FAS did not
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Fig. 4. Energy-filtering transmission electron microscopy
(EFTEM) on glycogen-located LBs. A: phosphorus-intensity
map of vesicular structures within glycogen generated by
EFTEM. The vesicular structures are enriched in phosphorus
(increased intensity depicted as white) as expected. Inset: electron micrograph of the same region labeled for CCT-␣ by
immunogold. b: TEM of an LB inside glycogen. c–e: intensity
maps of the same region using EFTEM for nitrogen (c) and
phosphorus (e). Pseudocolor overlay (d) of phosphorus (in red)
and nitrogen (in green). The central region of the glycogen
contains a low amount of both atoms, although there is a strong
phosphorus signal (e, arrows) coming from the edge of the
glycogen pool, which does not show in b. The LB is highly
enriched in phosphorus, as expected with a nominal amount of
protein. There is an intense nitrogen signal coming from one
region on the basal lateral side the LB (c and d, arrowheads).
Scale bar ⫽ 500 nm.
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SURFACTANT FORMATION IN FETAL LUNG CELLS
specifically localize to any visible structure, it appeared to
concentrate together in groups of 4 to 6 (Fig. 5A, solid circles).
The near absence of FAS from other cellular compartments at
a developmental stage of prominent de novo fatty acid synthesis (4, 24) suggests that FAS is functional within the glycogen
stores.
Finally, we examined the subcellular localization of surfactant proteins in the glycogen-laden type II cells. SP-A was
found to be absent from both the glycogen and intracellular
LBs. Rather, SP-A localized to the marginalized cytoplasm
(Fig. 5B). LBs in the alveolar space were found to be rich in
SP-A (Fig. 5C). These observations are consistent with SP-A
being secreted via a distinct pathway from surfactant lipids (28,
35, 48). One reason may be that if SP-A did enter the glycogenfilled regions of the cell it could become sequestered due to its
lectin properties and be unavailable for secretion. The exact
mechanism of SP-A secretion from the glycogen-filled type II
AJP-Lung Cell Mol Physiol • VOL
cells has not been elucidated. SP-B was primarily found within
LBs in the glycogen, although some of the gold label (5-nm
particles) could be observed to be independent of any lipid
structures (Fig. 5D). When 10-nm gold particles were used for
SP-B labeling, the identical ultrastructural distribution was
seen as with the 5-nm gold particles (data not shown). It is
unclear how SP-B reached the LBs within the glycogen, but
like FAS it tended to localize in clusters (Fig. 5D; circles).
Many SP-B (5 nm) gold particles within the LBs were in close
proximity of CCT-␣ (10 nm) gold particles (Fig. 5D). Because
SP-B is an essential structural component for LB formation
(37), the SP-B and CCT-␣ positive signals observed are indicators that LBs may be forming/maturing within the glycogen.
SP-C was also found within the glycogen, generally distributed
in clusters similar to FAS (Fig. 5E). Most of the protein was
not associated with any membrane structures. The pro-SP-Cpositive labels, which do not localize to any lipid structure,
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Fig. 5. Immunogold labeling for surfactant
related proteins in glycogen-filled type II
cells. Immunogold labeling for FAS (A);
almost all FAS label is concentrated in the
glycogen. Many of the gold particles are in
small groups (black circles). SP-A; intracellular (B) the label is found excluded from the
glycogen stores and distributed in the cytoplasm (white arrowheads). Extracellular (C)
secreted surfactant is intensely labeled for
SP-A (white arrowheads). D: SP-B; colabeling of CCT-␣ (10-nm gold particle, white
arrowheads) and SP-B (5-nm gold particle,
black circles) shows both proteins associating with the same vesicular structures inside
the glycogen. Also note the CCT-␣ bound to
ER (white asterisk). E: SP-C; colabeling of
CCT-␣ (10-nm gold particle, arrowhead)
and pro-SP-C (5-nm gold particle, black circles) shows both proteins inside the glycogen and at close proximity. Both SP-B and
SP-C seem to be congregated in tight groups
similar to FAS. No MVBs are visible. Scale
bar ⫽ 500 nm.
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SURFACTANT FORMATION IN FETAL LUNG CELLS
GRANTS
This work was supported by Canadian Institutes of Health Research Grant
FRN 36649. R. Ridsdale is a recipient of a Doctoral Research Award from the
Canadian Lung Association/Canadian Institutes of Health Research. M. Post is
the holder of a Canadian Research Chair (tier 1) in Fetal, Neonatal and
Maternal Health.
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