- Email: [email protected]
Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant
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
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 alveolar 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 interfacial 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 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.
Lamellar bodies are lysosome-related organelles
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 squamous 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 epithelium are cuboidal cells (type II cells) that form tight
Lamellar bodies range in size from 1.0 to 2.0 µM making the mature organelle one of the largest secretory 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 proteins (e.g., CD63/LAMP3 and LAMP1).5–8 Additional lysosomal characteristics of lamellar bodies include an acidic interior (pH ≈ 5.5) and extensive communication with the endocytic pathway.9–12 Lamellar bodies differ from lysosomes, but resemble a number of other lysosome-related organelles, in that they
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: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
263
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.
that inflation of the alveolus may be a physiologically relevant stimulus for surfactant secretion.21, 22 Measurements of surfactant secretion in isolated perfused lung further suggest that stretch-induced calcium oscillations in type I cells may also contribute to surfactant secretion by shuttling calcium to neighboring type II cells via gap junctions.23 In addition to stretch (i.e., mechano-transduction), stimulation of surfactant secretion through β-adrenergic, adenosine A2B and purinergic P2Y2 receptors has been described for cultured type II cells but the role of these pathways 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 fusion 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 aqueous extracellular environment may be very different from regulated exocytosis of secretory granules in endocrine and neuroendocrine cells; whether surfactant release in vivo is similarly regulated remains an important and unanswered question.
Packaging of surfactant in lamellar bodies Lamellar bodies are specialized for the storage of surfactant 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 phospholipid, 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 lamellar bodies.14–16 Pharmacological evidence for ATPdependent 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 facilitated 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 Intracellular signaling pathways leading to surfactant secretion have been extensively characterized in isolated type II cells.20 A key component of signaling 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 autoradiography 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.
SP-C
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 pathway for surfactant proteins. There is abundant electron 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 recycling pathways in type II epithelial cells to maintain surfactant homeostasis.
SP-C is a type II integral membrane protein that is expressed 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 lumenal domain. Processing of the proprotein in the multivesicular 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 intracellular packaging of surfactant since SP-C null mice have normal lamellar bodies;36 however, the mature peptide 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.
SP-B Mature SP-B isolated from the airspaces is a 79 residue peptide that co-extracts into organic solvents with surfactant phospholipids. SP-B is synthesized in type II epithelial cells as a 381 amino acid preproprotein in which the propeptide (residues 24–200) facilitates intracellular 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 vesicles 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 secreted 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 required both prenatally and postnatally for formation of mature lamellar bodies.
Trafficking of surfactant lipids to the lamellar body Newly secreted surfactant phospholipids, located in the alveolar hypophase, are inserted into the expanding 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 alveolar macrophages (a minor but important pathway in healthy subjects) or internalized by type II epithelial cells for recycling or degradation.39 Given the extreme hydrophobicity of SP-B and SP-C, it is highly likely that at least some surfactant phospholipid internalized from the alveolar airspace is routed with its associated surfactant proteins through the multivesicular body to the lamellar body. Whether recycling of surfactant phospholipid from the extracellular space 266
Lamellar body biogenesis
to the lamellar body occurs independently of the multivesicular body remains an open question. The multivesicular body may also be the source of lysobisphosphatidic acid, a minor phospholipid component of surfactant.13 The internal membranes of the multivesicular 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 surfactant 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 phosphatidylcholine 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 identified although several mechanisms for shuttling lipids from the ER to lamellar body have been proposed.
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, another member of the ABCA subclass, is expressed predominantly 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 phospholipids into lamellar bodies is autophagy. Autophagy is a pathway by which the cell incorporates parts of the cytoplasm or organelles into the lysosome for degradation 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 cytoplasm resulting in the formation of an autophagosome. Subsequent fusion of the outer membrane of the autophagosome with a lysosome leads to delivery of a membrane bound autophagic vesicle into the lumen of the lysosome. The delivery of cytoplasmic 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 detected 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 lamellar bodies, fetal type II epithelial cells contain large pools of glycogen in which disorganized or loosely organized strands of lipid that lack a limiting membrane 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
Phospholipid transfer proteins and ABC transporters Interorganelle transport of lipids can be accomplished by phospholipid transfer proteins. Proteins catalyzing the transfer of phosphatidylcholine, phosphatidylionositol 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 phosphatidylcholine transfer protein is highly specific for phosphatidylcholine, which comprises 70–80% of total phospholipid in surfactant; however, disruption of the locus encoding this transfer protein did not perturb lamellar body structure or alveolar surfactant 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
T.E. Weaver et al.
observations support a role for an autophagic-like process in lamellar body biogenesis, but direct evidence for transfer of surfactant phospholipids via this pathway 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 biogenesis of lamellar bodies or other lysosome-related organelles.
little progress in our understanding of lamellar body biogenesis. It is clear from the work of numerous investigators that the multivesicular body plays a key role in the delivery of newly synthesized surfactant proteins 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 hydrolases, LAMPs, and membrane proteins with putative transport functions, have not yet been established. A major unresolved issue in lamellar body maturation is the pathway(s) by which newly synthesized surfactant phospholipids are transported to and incorporated 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 Dietl 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 extracellular space followed by detachment from the plasma membrane (i.e., “run”) and retrieval to a more internal 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 essential step in assessing lamellar body turnover in type II cells. Finally, considerable insight into the key issues of surfactant secretion, transport and incorporation 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, basophil granules, and lamellar bodies) from normal mice and mice with genetic disorders would further enhance understanding of the biogenesis of these organelles.
Genetic disorders associated with altered lamellar body structure The biogenesis of lysosomes and lysosome-related organelles 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 significant complication of HPS. Morphological analyses of lung tissues from several HPS patients with interstitial 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 models 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 unresolved.
Acknowledgements Unresolved issues in lamellar body biogenesis
Research in the author’s laboratory (TEW) is supported by a MERIT award (R37-HL56285) and programmatic grants (PO1-HL56387 and PO1-HL61646) from the National Heart, Lung and Blood Institute.
Since the insightful autoradiographic study of Chevalier and Collet in 1972,27 there has been surprisingly 268
Lamellar body biogenesis
21. Wirtz HRW, Dobbs LG (1990) Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250:1266–1269 22. Tschumperlin DJ, Margulies SS (1999) Alveolar epithelial surface area–volume relationship in isolated rat lungs. J Appl Physiol 86:2026–2033 23. Ashino Y, Ying X, Dobbs LG, Bhattacharya J (2000) [Ca2+ ]i oscillations regulate type II cell exocytosis in the pulmonary alveolus. Am J Physiol 279:L5–L13 24. Rooney SA (2001) Regulation of surfactant secretion. Comp Biochem Physiol A Mol Integr Physiol 129:233–243 25. Haller T, Dietl P, Pfaller K, Frick M, Mair N, Paulmichl M, Hess MW, Furst J, Maly K (2001) Fusion pore expansion is a slow, discontinuous, and Ca2+ -dependent process regulating secretion from alveolar type II cells. J Cell Biol 155:279–289 26. Dietl P, Haller T (2000) Persistent fusion pores but transient fusion in alveolar type II cells. Cell Biol Int 24:803–807 27. Chevalier G, Collet AJ (1972) leucine-3H and galactose-3H in alveolar type II pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy. Anat Rec 174:289–310 28. Gobran LI, Rooney SA (2001) Regulation of SP-B and SP-C secretion in rat type II cells in primary culture. Am J Physiol 281:L1413–L1419 29. Voorhout WF, Weaver TE, Haagsman HP, Geuze HJ, van Golde LMJ (1993) Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells. Microsc Res Tech 26:366–373 30. Weaver TE, Conkright JJ (2001) Function of surfactant proteins B and C. Ann Rev Physiol 63:555–578 31. Weaver TE, Lin S, Bogucki B, Dey C (1992) Processing of surfactant protein-B proprotein by a cathepsin D-like protease. Am J Physiol 263:L95–L103 32. Hawgood S, Derrick M, Poulain F (1998) Structure and properties of surfactant protein B. Biochim Biophys Acta 1408: 150–160 33. Clark JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE, Whitsett JA (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad Sci USA 92:7794–7798 34. Nogee LM, DeMello DE, Dehner LP, Colten HR (1993) Deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N Engl J Med 328:406–410 35. Stahlman MT, Gray MP, Falconieri MW, Whitsett JA, Weaver TE (2000) Lamellar body formation in normal and surfactant protein B-deficient fetal mice. Lab Invest 80:395–403 36. Glasser SW, Burhans MS, Korfhagen TR, Na C-L, Sly PD, Ross GF, Ikegami M, Whitsett JA (2001) Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc Natl Acad Sci USA 98:6366–6371 37. Conkright JJ, Bridges JP, Na CL, Voorhout WF, Trapnell B, Glasser SW, Weaver TE (2001) Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide. J Biol Chem 276:14658–14664 38. Perez-Gil J, Keough KM (1998) Interfacial properties of surfactant proteins. Biochim Biophys Acta 1408:203–217 39. Wright JR (1990) Clearance and recycling of pulmonary surfactant. Am J Physiol 259:L1–L12 40. Kobayashi T, Stang E, Fang KS, de Moerloose P, Parton RG, Gruenberg J (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392:193–197
References 1. Schmitz G, Muller G (1991) Review: structure and function of lamellar bodies, lipid–protein complexes involved in storage and secretion of cellular lipids, lipid–protein complexes involved in storage and secretion of cellular lipids. J Lipid Res 32:1539–1570 2. Mason RJ, Shannon JM (1997) Alveolar type II cells, in The Lung: Scientific Foundations (Crystal RG, West JB, eds) pp. 543–555. Lippincott-Raven Publishers, Philadelphia, PA 3. Dietl P, Haller T, Mair N, Frick M (2001) Mechanisms of surfactant exocytosis in alveolar type II cells in vitro and in vivo. NIPS 16:239–243 4. Dell Angelica EC, Mullins C, Caplan S, Bonifacino JS (2000) Lysosome-related organelles. FASEB J 14:1265–1278 5. Yayoi Y, Ohsawa Y, Koike M, Zhang GQ, Kominami E, Uchiyama Y (2001) Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung. Arch Histol Cytol 64:89–97 6. Hook GE, Gilmore LB (1982) Hydrolases of pulmonary lysosomes and lamellar bodies. J Biol Chem 257:9211–9220 7. Wasano K, Hirakawa Y (1994) Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes. Histochemistry 102:329–335 8. Voorhout WF, Veenendaal T, Haagsman HP, Weaver TE, Whitsett JA, van Golde LMG, Geuze HJ (1992) Intracellular processing of pulmonary surfactant protein-B in an endosomal/lysosomal compartment. Am J Physiol 263:L479– L486 9. Williams MC (1984) Endocytosis in alveolar type II cells: effect of charge and size of tracers. Proc Natl Acad Sci USA 81:6054– 6058 10. Williams MC (1984) Uptake of lectins by pulmonary alveolar type II cells: subsequent deposition into lamellar bodies. Proc Natl Acad Sci USA 81:6383–6387 11. Young SL, Fram EK, Larson E, Wright JR (1993) Recycling of surfactant lipid and apoprotein-A studied by electron microscopic autoradiography. Am J Physiol 265:L19–L26 12. Kalina M, Socher R (1990) Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells. J Histochem Cytochem 38:483–492 13. Veldhuizen R, Nag K, Orgeig S, Possmayer F (1998) The role of lipids in pulmonary surfactant. Biochim Biophys Acta 1408:90– 108 14. Eckenhoff RG (1989) Perinatal changes in lung surfactant calcium measured in situ. J Clin Invest 84:1295–1301 15. Eckenhoff RG, Somlyo AP (1988) Rat lung type II cell and lamellar body: elemental composition in situ. Am J Physiol 254: C614–C620 16. Chinoy MR, Gonzales LW, Ballard PL, Fisher AB, Eckenhoff RG (1995) Elemental composition of lamellar bodies from fetal and adult human lung. Am J Respir Cell Mol Biol 13:99–108 17. Wadsworth SJ, Chander A (2000) H+ - and K+ -dependence of Ca2+ uptake in lung lamellar bodies. J Membr Biol 174:41–51 18. Wadsworth SJ, Spitzer AR, Chander A (1997) Ionic regulation of proton chemical (pH) and electrical gradients in lung lamellar bodies. Am J Physiol 17:L427–L436 19. Chander A, Sen N, Wadsworth S, Spitzer AR (2000) Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells. Lipids 35:35–43 20. Mason RJ, Voelker DR (1998) Regulatory mechanisms of surfactant secretion. Biochim Biophys Acta 1408:226–240
269
T.E. Weaver et al.
41. Osanai K, Mason RJ, Voelker DR (2001) Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells. Biochim Biophys Acta 1531:222–229 42. Lumb RH (1989) Phospholipid transfer proteins in mammalian lung. Am J Physiol 257:L190–L194 43. van Helvoort A, de Brouwer A, Ottenhoff R, Brouwers JFHM, Wijnholds J, Beijnen JH, Rijneveld A, vanderPoll T, van der Valk MA, Majoor D, Voorhout W, Wirtz KWA, Elferink RPJO, Borst P (1999) Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces. Proc Natl Acad Sci USA 96:11501–11506 44. McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, deWet J, Broccardo C, Chimini G, Francone OL (2000) High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci USA 97:4245–4250 45. Yamano G, Funahashi H, Kawanami O, Zhao LX, Ban N, Uchida Y, Morohoshi T, Ogawa J, Shioda S, Inagaki N (2001) ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells. FEBS Lett 508:221–225 46. Zen K, Notarfrancesco K, Oorschot V, Slot JW, Fisher AB, Shuman H (1998) Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane. Am J Physiol 19:L172–L183 47. Teter SA, Klionsky DJ (2000) Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation. Semin Cell Dev Biol 11:173–179 48. Thumm M (2000) Structure and function of the yeast vacuole and its role in autophagy. Microsc Res Tech 51:563–572 49. Hariri M, Millane G, Guimond MP, Guay G, Dennis JW, Nabi IR (2000) Biogenesis of multilamellar bodies via autophagy. Mol Biol Cell 11:255–268
50. Wattiez R, Hermans C, Bernard A, Lesur O, Falmagne P (1999) Human bronchoalveolar lavage fluid: two-dimensional gel electrophoresis, amino acid microsequencing and identification of major proteins. Electrophoresis 20:1634–1645 51. Huizing M, Anikster Y, Gahl WA (2000) Hermansky–Pudlak syndrome and related disorders of organelle formation. Traffic 1:823–835 52. Nakatani Y, Nakamura N, Sano J, Inayama Y, Kawano N, Yamanaka S, Miyagi Y, Nagashima Y, Ohbayashi C, Mizushima M, Manabe T, Kuroda M, Yokoi T, Matsubara O (2000) Interstitial pneumonia in Hermansky–Pudlak syndrome: significance of florid foamy swelling/degeneration (giant lamellar body degeneration) of type II pneumocytes. Virchows Archiv 437:304– 313 53. McGarry MP, Reddington M, Novak EK, Swank RT (1999) Survival and lung pathology of mouse models of Hermansky–Pudlak syndrome and Chediak–Higashi syndrome. Proc Soc Exp Biol Med 220:162–168 54. Swank RT, Novak EK, McGarry MP, Zhang YK, Li W, Zhang Q, Feng LJ (2000) Abnormal vesicular trafficking in mouse models of Hermansky–Pudlak syndrome. Pigm Cell Res 13:59–67 55. Chi EY, Prueitt JL, Lagunoff D (1975) Abnormal lamellar bodies in type II pneumocytes and increased lung surface active material in the beige mouse. J Histochem Cytochem 23:863– 869 56. Ward DM, Griffiths GM, Stinchcombe JC, Kaplan J (2000) Analysis of the lysosomal storage disease Chediak–Higashi syndrome. Traffic 1:816–822 57. Mullins C, Bonifacino JS (2001) The molecular machinery for lysosome biogenesis. Bioessays 23:333–343 58. Henkel AW, Kang G, Kornhuber J (2001) A common molecular machinery for exocytosis and the ‘kiss-and-run’ mechanism in chromaffin cells is controlled by phosphorylation. J Cell Sci 114:4613–4620
270
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
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 alveolar 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 interfacial 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 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.
Lamellar bodies are lysosome-related organelles
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 squamous 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 epithelium are cuboidal cells (type II cells) that form tight
Lamellar bodies range in size from 1.0 to 2.0 µM making the mature organelle one of the largest secretory 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 proteins (e.g., CD63/LAMP3 and LAMP1).5–8 Additional lysosomal characteristics of lamellar bodies include an acidic interior (pH ≈ 5.5) and extensive communication with the endocytic pathway.9–12 Lamellar bodies differ from lysosomes, but resemble a number of other lysosome-related organelles, in that they
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: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
263
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.
that inflation of the alveolus may be a physiologically relevant stimulus for surfactant secretion.21, 22 Measurements of surfactant secretion in isolated perfused lung further suggest that stretch-induced calcium oscillations in type I cells may also contribute to surfactant secretion by shuttling calcium to neighboring type II cells via gap junctions.23 In addition to stretch (i.e., mechano-transduction), stimulation of surfactant secretion through β-adrenergic, adenosine A2B and purinergic P2Y2 receptors has been described for cultured type II cells but the role of these pathways 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 fusion 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 aqueous extracellular environment may be very different from regulated exocytosis of secretory granules in endocrine and neuroendocrine cells; whether surfactant release in vivo is similarly regulated remains an important and unanswered question.
Packaging of surfactant in lamellar bodies Lamellar bodies are specialized for the storage of surfactant 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 phospholipid, 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 lamellar bodies.14–16 Pharmacological evidence for ATPdependent 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 facilitated 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 Intracellular signaling pathways leading to surfactant secretion have been extensively characterized in isolated type II cells.20 A key component of signaling 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 autoradiography 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.
SP-C
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 pathway for surfactant proteins. There is abundant electron 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 recycling pathways in type II epithelial cells to maintain surfactant homeostasis.
SP-C is a type II integral membrane protein that is expressed 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 lumenal domain. Processing of the proprotein in the multivesicular 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 intracellular packaging of surfactant since SP-C null mice have normal lamellar bodies;36 however, the mature peptide 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.
SP-B Mature SP-B isolated from the airspaces is a 79 residue peptide that co-extracts into organic solvents with surfactant phospholipids. SP-B is synthesized in type II epithelial cells as a 381 amino acid preproprotein in which the propeptide (residues 24–200) facilitates intracellular 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 vesicles 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 secreted 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 required both prenatally and postnatally for formation of mature lamellar bodies.
Trafficking of surfactant lipids to the lamellar body Newly secreted surfactant phospholipids, located in the alveolar hypophase, are inserted into the expanding 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 alveolar macrophages (a minor but important pathway in healthy subjects) or internalized by type II epithelial cells for recycling or degradation.39 Given the extreme hydrophobicity of SP-B and SP-C, it is highly likely that at least some surfactant phospholipid internalized from the alveolar airspace is routed with its associated surfactant proteins through the multivesicular body to the lamellar body. Whether recycling of surfactant phospholipid from the extracellular space 266
Lamellar body biogenesis
to the lamellar body occurs independently of the multivesicular body remains an open question. The multivesicular body may also be the source of lysobisphosphatidic acid, a minor phospholipid component of surfactant.13 The internal membranes of the multivesicular 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 surfactant 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 phosphatidylcholine 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 identified although several mechanisms for shuttling lipids from the ER to lamellar body have been proposed.
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, another member of the ABCA subclass, is expressed predominantly 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 phospholipids into lamellar bodies is autophagy. Autophagy is a pathway by which the cell incorporates parts of the cytoplasm or organelles into the lysosome for degradation 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 cytoplasm resulting in the formation of an autophagosome. Subsequent fusion of the outer membrane of the autophagosome with a lysosome leads to delivery of a membrane bound autophagic vesicle into the lumen of the lysosome. The delivery of cytoplasmic 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 detected 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 lamellar bodies, fetal type II epithelial cells contain large pools of glycogen in which disorganized or loosely organized strands of lipid that lack a limiting membrane 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
Phospholipid transfer proteins and ABC transporters Interorganelle transport of lipids can be accomplished by phospholipid transfer proteins. Proteins catalyzing the transfer of phosphatidylcholine, phosphatidylionositol 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 phosphatidylcholine transfer protein is highly specific for phosphatidylcholine, which comprises 70–80% of total phospholipid in surfactant; however, disruption of the locus encoding this transfer protein did not perturb lamellar body structure or alveolar surfactant 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
T.E. Weaver et al.
observations support a role for an autophagic-like process in lamellar body biogenesis, but direct evidence for transfer of surfactant phospholipids via this pathway 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 biogenesis of lamellar bodies or other lysosome-related organelles.
little progress in our understanding of lamellar body biogenesis. It is clear from the work of numerous investigators that the multivesicular body plays a key role in the delivery of newly synthesized surfactant proteins 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 hydrolases, LAMPs, and membrane proteins with putative transport functions, have not yet been established. A major unresolved issue in lamellar body maturation is the pathway(s) by which newly synthesized surfactant phospholipids are transported to and incorporated 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 Dietl 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 extracellular space followed by detachment from the plasma membrane (i.e., “run”) and retrieval to a more internal 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 essential step in assessing lamellar body turnover in type II cells. Finally, considerable insight into the key issues of surfactant secretion, transport and incorporation 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, basophil granules, and lamellar bodies) from normal mice and mice with genetic disorders would further enhance understanding of the biogenesis of these organelles.
Genetic disorders associated with altered lamellar body structure The biogenesis of lysosomes and lysosome-related organelles 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 significant complication of HPS. Morphological analyses of lung tissues from several HPS patients with interstitial 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 models 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 unresolved.
Acknowledgements Unresolved issues in lamellar body biogenesis
Research in the author’s laboratory (TEW) is supported by a MERIT award (R37-HL56285) and programmatic grants (PO1-HL56387 and PO1-HL61646) from the National Heart, Lung and Blood Institute.
Since the insightful autoradiographic study of Chevalier and Collet in 1972,27 there has been surprisingly 268
Lamellar body biogenesis
21. Wirtz HRW, Dobbs LG (1990) Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250:1266–1269 22. Tschumperlin DJ, Margulies SS (1999) Alveolar epithelial surface area–volume relationship in isolated rat lungs. J Appl Physiol 86:2026–2033 23. Ashino Y, Ying X, Dobbs LG, Bhattacharya J (2000) [Ca2+ ]i oscillations regulate type II cell exocytosis in the pulmonary alveolus. Am J Physiol 279:L5–L13 24. Rooney SA (2001) Regulation of surfactant secretion. Comp Biochem Physiol A Mol Integr Physiol 129:233–243 25. Haller T, Dietl P, Pfaller K, Frick M, Mair N, Paulmichl M, Hess MW, Furst J, Maly K (2001) Fusion pore expansion is a slow, discontinuous, and Ca2+ -dependent process regulating secretion from alveolar type II cells. J Cell Biol 155:279–289 26. Dietl P, Haller T (2000) Persistent fusion pores but transient fusion in alveolar type II cells. Cell Biol Int 24:803–807 27. Chevalier G, Collet AJ (1972) leucine-3H and galactose-3H in alveolar type II pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy. Anat Rec 174:289–310 28. Gobran LI, Rooney SA (2001) Regulation of SP-B and SP-C secretion in rat type II cells in primary culture. Am J Physiol 281:L1413–L1419 29. Voorhout WF, Weaver TE, Haagsman HP, Geuze HJ, van Golde LMJ (1993) Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells. Microsc Res Tech 26:366–373 30. Weaver TE, Conkright JJ (2001) Function of surfactant proteins B and C. Ann Rev Physiol 63:555–578 31. Weaver TE, Lin S, Bogucki B, Dey C (1992) Processing of surfactant protein-B proprotein by a cathepsin D-like protease. Am J Physiol 263:L95–L103 32. Hawgood S, Derrick M, Poulain F (1998) Structure and properties of surfactant protein B. Biochim Biophys Acta 1408: 150–160 33. Clark JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE, Whitsett JA (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad Sci USA 92:7794–7798 34. Nogee LM, DeMello DE, Dehner LP, Colten HR (1993) Deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N Engl J Med 328:406–410 35. Stahlman MT, Gray MP, Falconieri MW, Whitsett JA, Weaver TE (2000) Lamellar body formation in normal and surfactant protein B-deficient fetal mice. Lab Invest 80:395–403 36. Glasser SW, Burhans MS, Korfhagen TR, Na C-L, Sly PD, Ross GF, Ikegami M, Whitsett JA (2001) Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc Natl Acad Sci USA 98:6366–6371 37. Conkright JJ, Bridges JP, Na CL, Voorhout WF, Trapnell B, Glasser SW, Weaver TE (2001) Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide. J Biol Chem 276:14658–14664 38. Perez-Gil J, Keough KM (1998) Interfacial properties of surfactant proteins. Biochim Biophys Acta 1408:203–217 39. Wright JR (1990) Clearance and recycling of pulmonary surfactant. Am J Physiol 259:L1–L12 40. Kobayashi T, Stang E, Fang KS, de Moerloose P, Parton RG, Gruenberg J (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392:193–197
References 1. Schmitz G, Muller G (1991) Review: structure and function of lamellar bodies, lipid–protein complexes involved in storage and secretion of cellular lipids, lipid–protein complexes involved in storage and secretion of cellular lipids. J Lipid Res 32:1539–1570 2. Mason RJ, Shannon JM (1997) Alveolar type II cells, in The Lung: Scientific Foundations (Crystal RG, West JB, eds) pp. 543–555. Lippincott-Raven Publishers, Philadelphia, PA 3. Dietl P, Haller T, Mair N, Frick M (2001) Mechanisms of surfactant exocytosis in alveolar type II cells in vitro and in vivo. NIPS 16:239–243 4. Dell Angelica EC, Mullins C, Caplan S, Bonifacino JS (2000) Lysosome-related organelles. FASEB J 14:1265–1278 5. Yayoi Y, Ohsawa Y, Koike M, Zhang GQ, Kominami E, Uchiyama Y (2001) Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung. Arch Histol Cytol 64:89–97 6. Hook GE, Gilmore LB (1982) Hydrolases of pulmonary lysosomes and lamellar bodies. J Biol Chem 257:9211–9220 7. Wasano K, Hirakawa Y (1994) Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes. Histochemistry 102:329–335 8. Voorhout WF, Veenendaal T, Haagsman HP, Weaver TE, Whitsett JA, van Golde LMG, Geuze HJ (1992) Intracellular processing of pulmonary surfactant protein-B in an endosomal/lysosomal compartment. Am J Physiol 263:L479– L486 9. Williams MC (1984) Endocytosis in alveolar type II cells: effect of charge and size of tracers. Proc Natl Acad Sci USA 81:6054– 6058 10. Williams MC (1984) Uptake of lectins by pulmonary alveolar type II cells: subsequent deposition into lamellar bodies. Proc Natl Acad Sci USA 81:6383–6387 11. Young SL, Fram EK, Larson E, Wright JR (1993) Recycling of surfactant lipid and apoprotein-A studied by electron microscopic autoradiography. Am J Physiol 265:L19–L26 12. Kalina M, Socher R (1990) Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells. J Histochem Cytochem 38:483–492 13. Veldhuizen R, Nag K, Orgeig S, Possmayer F (1998) The role of lipids in pulmonary surfactant. Biochim Biophys Acta 1408:90– 108 14. Eckenhoff RG (1989) Perinatal changes in lung surfactant calcium measured in situ. J Clin Invest 84:1295–1301 15. Eckenhoff RG, Somlyo AP (1988) Rat lung type II cell and lamellar body: elemental composition in situ. Am J Physiol 254: C614–C620 16. Chinoy MR, Gonzales LW, Ballard PL, Fisher AB, Eckenhoff RG (1995) Elemental composition of lamellar bodies from fetal and adult human lung. Am J Respir Cell Mol Biol 13:99–108 17. Wadsworth SJ, Chander A (2000) H+ - and K+ -dependence of Ca2+ uptake in lung lamellar bodies. J Membr Biol 174:41–51 18. Wadsworth SJ, Spitzer AR, Chander A (1997) Ionic regulation of proton chemical (pH) and electrical gradients in lung lamellar bodies. Am J Physiol 17:L427–L436 19. Chander A, Sen N, Wadsworth S, Spitzer AR (2000) Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells. Lipids 35:35–43 20. Mason RJ, Voelker DR (1998) Regulatory mechanisms of surfactant secretion. Biochim Biophys Acta 1408:226–240
269
T.E. Weaver et al.
41. Osanai K, Mason RJ, Voelker DR (2001) Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells. Biochim Biophys Acta 1531:222–229 42. Lumb RH (1989) Phospholipid transfer proteins in mammalian lung. Am J Physiol 257:L190–L194 43. van Helvoort A, de Brouwer A, Ottenhoff R, Brouwers JFHM, Wijnholds J, Beijnen JH, Rijneveld A, vanderPoll T, van der Valk MA, Majoor D, Voorhout W, Wirtz KWA, Elferink RPJO, Borst P (1999) Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces. Proc Natl Acad Sci USA 96:11501–11506 44. McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, deWet J, Broccardo C, Chimini G, Francone OL (2000) High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci USA 97:4245–4250 45. Yamano G, Funahashi H, Kawanami O, Zhao LX, Ban N, Uchida Y, Morohoshi T, Ogawa J, Shioda S, Inagaki N (2001) ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells. FEBS Lett 508:221–225 46. Zen K, Notarfrancesco K, Oorschot V, Slot JW, Fisher AB, Shuman H (1998) Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane. Am J Physiol 19:L172–L183 47. Teter SA, Klionsky DJ (2000) Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation. Semin Cell Dev Biol 11:173–179 48. Thumm M (2000) Structure and function of the yeast vacuole and its role in autophagy. Microsc Res Tech 51:563–572 49. Hariri M, Millane G, Guimond MP, Guay G, Dennis JW, Nabi IR (2000) Biogenesis of multilamellar bodies via autophagy. Mol Biol Cell 11:255–268
50. Wattiez R, Hermans C, Bernard A, Lesur O, Falmagne P (1999) Human bronchoalveolar lavage fluid: two-dimensional gel electrophoresis, amino acid microsequencing and identification of major proteins. Electrophoresis 20:1634–1645 51. Huizing M, Anikster Y, Gahl WA (2000) Hermansky–Pudlak syndrome and related disorders of organelle formation. Traffic 1:823–835 52. Nakatani Y, Nakamura N, Sano J, Inayama Y, Kawano N, Yamanaka S, Miyagi Y, Nagashima Y, Ohbayashi C, Mizushima M, Manabe T, Kuroda M, Yokoi T, Matsubara O (2000) Interstitial pneumonia in Hermansky–Pudlak syndrome: significance of florid foamy swelling/degeneration (giant lamellar body degeneration) of type II pneumocytes. Virchows Archiv 437:304– 313 53. McGarry MP, Reddington M, Novak EK, Swank RT (1999) Survival and lung pathology of mouse models of Hermansky–Pudlak syndrome and Chediak–Higashi syndrome. Proc Soc Exp Biol Med 220:162–168 54. Swank RT, Novak EK, McGarry MP, Zhang YK, Li W, Zhang Q, Feng LJ (2000) Abnormal vesicular trafficking in mouse models of Hermansky–Pudlak syndrome. Pigm Cell Res 13:59–67 55. Chi EY, Prueitt JL, Lagunoff D (1975) Abnormal lamellar bodies in type II pneumocytes and increased lung surface active material in the beige mouse. J Histochem Cytochem 23:863– 869 56. Ward DM, Griffiths GM, Stinchcombe JC, Kaplan J (2000) Analysis of the lysosomal storage disease Chediak–Higashi syndrome. Traffic 1:816–822 57. Mullins C, Bonifacino JS (2001) The molecular machinery for lysosome biogenesis. Bioessays 23:333–343 58. Henkel AW, Kang G, Kornhuber J (2001) A common molecular machinery for exocytosis and the ‘kiss-and-run’ mechanism in chromaffin cells is controlled by phosphorylation. J Cell Sci 114:4613–4620
270