seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp.
263–270
doi:10.1016/S1084–9521(02)00055-1, available online at http://www.idealibrary.com on
Biogenesis of lamellar bodies, lysosome-related organelles
involved in storage and secretion of pulmonary surfactant
Timothy E. Weaver a,∗ , Cheng-Lun Na a and Mildred Stahlman b
Lamellar bodies are members of a subclass of lysosome-related
organelles referred to as secretory lysosomes. The principal
constituents of the lamellar body, surfactant phospholipids,
are organized into tightly packed, bilayer membranes in
a process that is strongly influenced by the lung-specific,
hydrophobic peptide SP-B. Newly synthesized SP-B is
transported from the Golgi to the lamellar body via
multivesicular bodies; in contrast, preliminary evidence
suggests that newly synthesized surfactant phospholipids are
transported from the ER and incorporated into the internal
membranes of the lamellar body via a distinct pathway.
Abbreviations: ER, endoplasmic reticulum / LB, lamellar
body / MIT, mitochondrion / MVB, multivesicular body /
NUC, nucleus
Key words: autophagy / exocytosis / multivesicular body /
phospholipid transfer protein / secretory granules
© 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Pulmonary alveoli are the terminal sac-like extensions
of the distal respiratory tree that are specialized for
gas exchange. The alveolar surface is covered by squa-
mous epithelial cells (type I cells) that, together with
the underlying basement membrane and capillary
endothelium, comprise 90% of the air–blood barrier
in the lung. Interspersed along the alveolar epithe-
lium are cuboidal cells (type II cells) that form tight
From the a Division of Pulmonary Biology, Cincinnati Children’s Hospital
Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
and b Division of Neonatology, Vanderbilt University School of Medicine,
21st & Garland, Nashville, TN 37232-2370, USA. * Corresponding
author. E-mail: tim.weaver@chmcc.org
© 2002 Elsevier Science Ltd. All rights reserved.
1084–9521 / 02 / $– see front matter
263
junctions with type I cells to maintain a selectively
permeable epithelial barrier. Alveoli are inherently
unstable structures due in large part to a thin liquid
lining layer (hypophase) that generates high surface
tension forces as alveolar diameter narrows during
expiration. Elevated surface tension can lead to alve-
olar collapse, making it very difficult to expand the
alveolus during the subsequent inspiratory cycle and,
ultimately, resulting in respiratory distress syndrome
and the need for ventilatory support. This highly
undesirable outcome is prevented by formation of
a phospholipid film at the air–liquid interface that
dramatically reduces surface tension as the interfa-
cial film is compressed during expiration. The lipid
film (pulmonary surfactant) is synthesized in type
II epithelial cells and stored in specialized secretory
granules prior to secretion into the hypophase. These
secretory granules, referred to as lamellar bodies,
are detected in a wide range of tissues;1 this review
will focus exclusively to lamellar bodies in pulmonary
type II epithelial cells.
Lamellar bodies are lysosome-related
organelles
Lamellar bodies range in size from 1.0 to 2.0 µM
making the mature organelle one of the largest se-
cretory granules in any cell type.2, 3 The lysosomal
nature of lamellar bodies has long been appreciated
by workers in the lung field but largely overlooked
in studies of lysosome-related organelles.4 Similar
to lysosomes and many lysosome-related organelles,
lamellar bodies contain soluble lysosomal enzymes
(e.g., acid phosphatase, cathepsins C and H) and pro-
teins (e.g., CD63/LAMP3 and LAMP1).5–8 Additional
lysosomal characteristics of lamellar bodies include
an acidic interior (pH ≈ 5.5) and extensive commu-
nication with the endocytic pathway.9–12 Lamellar
bodies differ from lysosomes, but resemble a num-
ber of other lysosome-related organelles, in that they
T.E. Weaver et al.

Figure 1. Lamellar bodies at various stages of development in type II cells of fetal mouse lung. Well-developed multivesicular
bodies and particulate glycogen (arrows) are characteristic of this stage of type II cell differentiation. Scale bar: 500 nm.

Figure 2. Fusion of a multivesicular body with a lamellar body in a fetal mouse type II cell. The limiting membrane (arrow)
of the lamellar body is continuous with that of the multivesicular body. Scale bar: 200 nm. Reproduced from.35

Figure 3. Lamellar body formation in SP-B (−/−) mice. Typical multivesicular bodies are detected in type II cells of SP-B
(−/−) fetal lung. Lamellar bodies contain some peripheral membrane lamellae (arrow heads) but consist primarily of small
vesicles (large arrows) similar to those in mature multivesicular bodies. Note glycogen particles (small arrow) trapped inside
the disorganized lamellar body. Scale bar: 500 nm.

264
 Lamellar body biogenesis

are specialized for storage and secretion rather than
degradation of their contents.
Packaging of surfactant in lamellar bodies
Lamellar bodies are specialized for the storage of sur-
factant phospholipids which are arranged in the form
of tightly packed, concentric, membrane sheets or
lamellae (Figures 1 and 2). The bilayer membranes are
highly enriched in dipalmitoylphosphatidylcholine,
the major surface tension reducing phospholipid in
the extracellular surfactant film.13 Lamellar bodies
also contain a significant proportion of acidic phos-
pholipid, principally phosphatidylglycerol, which
could potentially disrupt membrane packing via
charge repulsion. This problem may be overcome in
part by sequestering high levels of calcium in lamel-
lar bodies.14–16 Pharmacological evidence for ATP-
dependent uptake of calcium into isolated lamellar
bodies has been reported but the molecular identify
of the pump(s) has not been determined.17 Both
calcium uptake and phospholipid packaging are fa-
cilitated by an acidic lumenal environment which
is maintained by a vacuolar type H+ -ATPase.17–19
An additional critical component of phospholipid
packaging is the incorporation of small intralumenal
vesicles, derived from fusion of multivesicular bodies
with the lamellar body, into the membrane sheets;
the reorganization of intravesicular lipid structure is
regulated in part by the surfactant-associated protein
SP-B (discussed below).
Release of surfactant from lamellar bodies
that inflation of the alveolus may be a physiologically
relevant stimulus for surfactant secretion.21, 22 Mea-
surements of surfactant secretion in isolated perfused
lung further suggest that stretch-induced calcium
oscillations in type I cells may also contribute to sur-
factant secretion by shuttling calcium to neighboring
type II cells via gap junctions.23 In addition to stretch
(i.e., mechano-transduction), stimulation of surfac-
tant secretion through β-adrenergic, adenosine A2B
and purinergic P2Y2 receptors has been described
for cultured type II cells but the role of these path-
ways in modulating surfactant secretion in vivo is less
certain.24 Secretagogue stimulation of type II cells in
culture results in rapid formation of a fusion pore
between the lamellar body and plasma membrane
followed by a much slower and prolonged release of
surfactant from the lamellar body.25 Modulation of fu-
sion pore size, and consequently surfactant release, is
also calcium-dependent providing an additional level
of regulation of surfactant secretion. Interestingly,
there is preliminary evidence that following discharge
of some of its surfactant cargo, the lamellar body is
released from the cell surface and moves to a more
internal location;26 the latter observation, if true, has
important implications for lamellar body biogenesis
(discussed below). Collectively, the results of in vitro
studies imply that stimulated secretion and dispersion
of the hydrophobic surfactant complex into an aque-
ous extracellular environment may be very different
from regulated exocytosis of secretory granules in
endocrine and neuroendocrine cells; whether surfac-
tant release in vivo is similarly regulated remains an
important and unanswered question.

Intracellular signaling pathways leading to surfac-

tant secretion have been extensively characterized
in isolated type II cells.20 A key component of sig-
naling involves the elevation of cytoplasmic calcium
levels which has been shown to occur in response
to cell stretch (as well as other stimuli), suggesting
Trafficking of surfactant proteins to the
lamellar body
The results of an electron microscopic autoradiog-
raphy study with 3 [H] leucine established that newly

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 4. Localization of SP-C to internal vesicles of the multivesicular body. Ultrathin cryosections from fetal mouse lung
were incubated with antibody directed against the cytosolic domain of the SP-C proprotein. Gold particles were detected
on the limiting membrane of the multivesicular body and within the internal vesicles (arrows) of this organelle. The lipid
contents of lamellar bodies were extracted during sample preparation. Scale bar: 200 nm. Reproduced from.37

Figure 5. Primitive membrane systems in immature fetal mouse type II cells. Loosely organized membranes (arrow) in
a pool of glycogen are frequently detected prior to the appearance of typical lamellar bodies in fetal type II cells. Direct
evidence that these membranes are in an early stage in the formation of lamellar bodies is still lacking. Scale bar: 500 nm.

Figure 6. Glycogen particles (arrow) trapped inside an immature lamellar body from a fetal mouse type II cell. Scale bar:
100 nm.

265
T.E. Weaver et al.
synthesized protein in type II cells was sequentially
detected in the ER, Golgi and multivesicular bodies,
and lamellar bodies.27 SP-B and surfactant protein
C (SP-C), lung-specific proteins that are co-secreted
with surfactant phospholipids,28 were localized by
immunogold labeling to the same intracellular
compartments,29 consistent with the hypothesis that
these elements comprise the major biosynthetic path-
way for surfactant proteins. There is abundant elec-
tron microscope evidence for fusion of multivesicular
bodies with lamellar bodies, suggesting that this event
represents the final stage in the delivery of newly
synthesized proteins to the lamellar body (Figure 2).
Turnover of surface film components in the alveolar
airspace results in the internalization of SP-B and SP-C
into the endocytic pathway of type II epithelial cells
and recycling of these peptides to the lamellar body
via a pathway that traverses the multivesicular body. It
is therefore likely that the multivesicular body plays a
critical role in integrating the biosynthetic and recy-
cling pathways in type II epithelial cells to maintain
surfactant homeostasis.
SP-B
Mature SP-B isolated from the airspaces is a 79 residue
peptide that co-extracts into organic solvents with sur-
factant phospholipids. SP-B is synthesized in type II
epithelial cells as a 381 amino acid preproprotein in
which the propeptide (residues 24–200) facilitates in-
tracellular trafficking of the extremely hydrophobic
mature peptide (residues 201–279).30 Processing of
the proprotein to the mature peptide occurs in the
multivesicular body,8, 31 resulting in the association of
the ampiphathic helices of the mature peptide with
lumenal vesicles. The fusogenic and lytic properties32
of the newly liberated mature peptide likely play an
important role in the incorporation of the internal
vesicles of multivesicular bodies into the concentric
membranes of lamellar bodies following the fusion
of these two organelles (Figure 2). In the absence of
SP-B, lamellar bodies contain numerous internal vesi-
cles with few membrane lamellae, consistent with an
essential role for SP-B in the packaging of surfactant
phospholipids in lamellar bodies33 (Figure 3). The
contents of these disorganized lamellar bodies are se-
creted into the alveolar airspace, but fail to form a
functional surfactant film resulting in alveolar collapse
and, ultimately, death.34, 35 SP-B is therefore absolutely
required for postnatal lung function and is also re-
quired both prenatally and postnatally for formation
of mature lamellar bodies.
266
SP-C
SP-C is a type II integral membrane protein that is ex-
pressed exclusively in alveolar type II epithelial cells
of the lung.30 The 197 amino acid SP-C proprotein is
composed of a short (35 amino acids) cytosolic tail, a
transmembrane domain, and a relatively large lume-
nal domain. Processing of the proprotein in the multi-
vesicular body/lamellar body results in generation of
the mature peptide, consisting of the transmembrane
domain and 12 amino acids of the cytosolic domain.
The mature SP-C peptide is not involved in intracellu-
lar packaging of surfactant since SP-C null mice have
normal lamellar bodies;36 however, the mature pep-
tide is secreted into the alveolar space with surfactant
phospholipids where it plays an important role in the
formation and maintenance of the surfactant film. A
key event in the secretion of this integral membrane
peptide is the inward vesiculation of the limiting
membrane of the multivesicular body which results in
relocation of the proprotein from the surface of the
organelle to small intralumenal vesicles (Figure 4).
The cytosolic domain of the proprotein is required
for secretion of SP-C, but the molecular machinery
that interacts with this domain to direct sorting to
and internalization at the multivesicular body has not
been identified.37 Fusion of the multivesicular body
with the lamellar body results in transfer of the SP-C
containing lumenal vesicles to the lamellar body;
SP-B-mediated incorporation of these vesicles into
the internal membranes of the lamellar body results
in co-secretion of SP-C with surfactant phospholipids.
Trafficking of surfactant lipids
to the lamellar body
Newly secreted surfactant phospholipids, located in
the alveolar hypophase, are inserted into the expand-
ing surface film in a poorly understood process that is
dependent on SP-B and SP-C.38 Repeated expansion
and contraction of the surface film leads to loss of
film components which are either cleared by alveo-
lar macrophages (a minor but important pathway in
healthy subjects) or internalized by type II epithelial
cells for recycling or degradation.39 Given the ex-
treme hydrophobicity of SP-B and SP-C, it is highly
likely that at least some surfactant phospholipid in-
ternalized from the alveolar airspace is routed with its
associated surfactant proteins through the multivesic-
ular body to the lamellar body. Whether recycling of
surfactant phospholipid from the extracellular space

to the lamellar body occurs independently of the
multivesicular body remains an open question. The
multivesicular body may also be the source of lysobis-
phosphatidic acid, a minor phospholipid component
of surfactant.13 The internal membranes of the mul-
tivesicular body are enriched in this lipid40 which
is likely transferred to the lamellar body following
fusion of these organelles.
Although the multivesicular body almost certainly
plays some role in the recycling of extracellular sur-
factant phospholipids to the lamellar body, at least
two lines of evidence suggest that newly synthesized
surfactant phospholipids follow a different pathway
to the lamellar body. First, treatment of type II cells in
culture with brefeldin A completely blocked transport
of newly synthesized protein through the Golgi to the
lamellar body, but did not alter phosphatidylcholine
transport to the lamellar body.41 Second, an electron
microscopic autoradiography study with 3 [H]-choline
detected label in the ER, Golgi and lamellar bodies,
but not multivesicular bodies;27 label localized to
the Golgi may well have been due to phosphatidyl-
choline destined for intracellular membranes other
than the lamellar body. These data are consistent with
a novel phospholipid transport pathway from the ER,
the site of surfactant phospholipid synthesis, to the
lamellar body. This pathway has not yet been identi-
fied although several mechanisms for shuttling lipids
from the ER to lamellar body have been proposed.
Phospholipid transfer proteins and ABC transporters
Interorganelle transport of lipids can be accom-
plished by phospholipid transfer proteins. Proteins
catalyzing the transfer of phosphatidylcholine, phos-
phatidylionositol and phosphatidylglycerol have been
identified in the lung, and their transfer properties
have been characterized in vitro;42 however, to date,
there is no direct evidence that any of these proteins
play an important role in the movement of surfactant
lipids from the ER to the lamellar body. The phos-
phatidylcholine transfer protein is highly specific for
phosphatidylcholine, which comprises 70–80% of to-
tal phospholipid in surfactant; however, disruption
of the locus encoding this transfer protein did not
perturb lamellar body structure or alveolar surfac-
tant composition.43 It is possible that upregulation
of non-specific or novel-specific transfer proteins
compensated for loss of phosphatidylcholine transfer
protein.
Transport of surfactant phospholipids across the
lamellar body membrane could be facilitated by one
267
Lamellar body biogenesis
or more members of the ATP-binding cassette (ABC)
transporter superfamily, in particular members of the
ABCA subclass, several of which have been implicated
in lipid transport. Disruption of the locus encoding
ABCA1 resulted in the accumulation of lipid within
alveolar macrophages and type II cells.44 ABCA2, an-
other member of the ABCA subclass, is expressed pre-
dominantly in the lung and appears to be enriched
in type II cells where it is localized to the limiting
membranes of lamellar bodies.45, 46 Taken together,
these data suggest that one possible mechanism for
incorporation of surfactant phospholipids into the
lamellar body is transport from the ER to the lamellar
body by a phospholipid transfer protein(s) followed
by translocation into the lumen of the lamellar body
by an ABCA protein(s).
Autophagy
An alternative (but not necessarily mutually exclusive)
mechanism for incorporation of surfactant phospho-
lipids into lamellar bodies is autophagy. Autophagy is
a pathway by which the cell incorporates parts of the
cytoplasm or organelles into the lysosome for degra-
dation during nutrient deprivation.47, 48 The process
is initiated by the formation of a double-membrane
structure that grows to enclose a region of the cyto-
plasm resulting in the formation of an autophago-
some. Subsequent fusion of the outer membrane of
the autophagosome with a lysosome leads to deliv-
ery of a membrane bound autophagic vesicle into
the lumen of the lysosome. The delivery of cytoplas-
mic components into lamellar body-like structures in
a transformed alveolar epithelial cell line has been
reported;49 incorporation into the lamellar body was
reversibly inhibited by 3-methyladenine, an inhibitor
of autophagy. Interestingly at least one cytoplasmic
protein, triose phosphate isomerase, has been de-
tected in bronchoalveolar lavage fluid,50 consistent
with incorporation into a secretory granule prior to
secretion. Further evidence supporting incorporation
of cytoplasmic elements into lamellar bodies comes
from ultrastructural studies of type II epithelial cells
in fetal lung.35 Prior to formation of mature lamel-
lar bodies, fetal type II epithelial cells contain large
pools of glycogen in which disorganized or loosely
organized strands of lipid that lack a limiting mem-
brane are frequently detected (Figure 5). As type
II epithelial cell differentiation proceeds, glycogen
pools are depleted and lamellar bodies with intact
limiting membranes appear: Some of these lamellar
bodies contain glycogen granules (Figure 6). These
T.E. Weaver et al.
observations support a role for an autophagic-like pro-
cess in lamellar body biogenesis, but direct evidence
for transfer of surfactant phospholipids via this path-
way is still lacking. Since many of the genes involved
in autophagy and the related cytoplasm-to-vacuole
pathway have been identified in yeast, it should soon
be possible to directly determine if this degradative
pathway also plays an important role in the biogen-
esis of lamellar bodies or other lysosome-related
organelles.
Genetic disorders associated with altered
lamellar body structure
The biogenesis of lysosomes and lysosome-related or-
ganelles are affected in Chediak–Higashi syndrome
(CHS) and Hermansky–Pudlak syndrome (HPS)
leading to pigmentation and bleeding disorders.51
Interestingly, fibrotic lung disease is also a signifi-
cant complication of HPS. Morphological analyses
of lung tissues from several HPS patients with inter-
stitial lung disease detected giant lamellar bodies in
type II epithelial cells as well as patchy fibrosis and
enlargement of airspaces.52 Altered lung structure
and survival was also detected in several mouse mod-
els of HPS although it is not yet clear if lamellar
body size/structure is affected in these animals.53
The genes affected in each of the mouse mutants
are involved at some level in vesicle formation or
trafficking.54 Greatly enlarged lamellar bodies were
also detected in beige mice,55 a model of CHS which
is characterized by giant lysosomes in multiple tissues.
The Beige/LYST gene product has been implicated
in fusion/fission events that regulate lysosome size.56
Lung disease is not typically associated with CHS but
this may be related to the significantly shorter life span
of CHS patients compared to HPS patients. Overall,
the observation that genetic mutations underlying
CHS and HPS, alter the phenotype of lamellar bodies
as well as that of lysosomes, platelet dense granules
and melanosomes strongly supports classification of
the lamellar body as a lysosome-related organelle;
however, the precise roles that these genes play in
biogenesis of lysosome-related organelles remain un-
resolved.
Unresolved issues in lamellar body biogenesis
Since the insightful autoradiographic study of Cheva-
lier and Collet in 1972,27 there has been surprisingly
268
little progress in our understanding of lamellar body
biogenesis. It is clear from the work of numerous in-
vestigators that the multivesicular body plays a key role
in the delivery of newly synthesized surfactant pro-
teins as well as peptides internalized via the endocytic
pathway (with their accompanying phospholipids)
to the lamellar body. One of these peptides, SP-B, is
essential for organization of surfactant phospholipids
in the lamellar body. The roles of other lamellar body
proteins, including multiple acid-dependent hydro-
lases, LAMPs, and membrane proteins with putative
transport functions, have not yet been established.
A major unresolved issue in lamellar body matura-
tion is the pathway(s) by which newly synthesized
surfactant phospholipids are transported to and in-
corporated into the internal membrane lamellae.
The mechanism by which the lumenal contents of
the lamellar body are subsequently released into the
airspace will also impact biogenesis of this organelle.
The model of surfactant secretion proposed by Di-
etl et al.3 is similar to the “kiss-and-run” model of
lysosome maturation.57, 58 A “prolonged kiss” (i.e.,
fusion of the limiting membrane of a lamellar body
with the plasma membrane to form a fusion pore)
allows slow release of surfactant into the extracellu-
lar space followed by detachment from the plasma
membrane (i.e., “run”) and retrieval to a more in-
ternal location in the cell: since the lamellar body
remains intact, the need to generate a new organelle
is correspondingly reduced. A better understanding
of the potential contribution of this pathway and/or
classical exocytosis to surfactant secretion is an es-
sential step in assessing lamellar body turnover in
type II cells. Finally, considerable insight into the key
issues of surfactant secretion, transport and incor-
poration of surfactant lipids into the lamellar body,
and targeting of proteins to this organelle would
come from identifying the lamellar body proteome.
Systematic comparison of proteomes for secretory
lysosomes (lytic granules, platelet dense granules, ba-
sophil granules, and lamellar bodies) from normal
mice and mice with genetic disorders would further
enhance understanding of the biogenesis of these
organelles.
Acknowledgements
Research in the author’s laboratory (TEW) is sup-
ported by a MERIT award (R37-HL56285) and pro-
grammatic grants (PO1-HL56387 and PO1-HL61646)
from the National Heart, Lung and Blood Institute.

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